Analysis of preservatives in pharmaceutical products

Khairi M.S. Fahelelbom

Preservatives are substances that commonly added to various foods and pharmaceutical products in order to prolong their shelf life. The addition of preservatives to such products, especially to those that have higher water content, is essential for avoiding alteration and degradation by microorganisms during storage ^1-3 .

Other ingredients are also utilized in preparing the desired dosage form of a drug substance. Some of these agents may be used to achieve the desired physical and chemical characteristics of the product or to improve its appearance, odor and taste. In each instant, the added ingredient must be harmless in the amount used; does not exceed the minimum quantity required to provide its intended effect; its presence does not impair the bioavailability, the therapeutic efficacy or safety of the official preparation, and does not interfere with analysis and tests prescribed for determining compliance with the pharmacopeias standards ⁴ .

II. Classification of preservatives

Preservatives are classified into two main classes: antimicrobial preservatives and antioxidants ^1-3 .

II. A. Antimicrobial preservatives:

Antimicrobial preservatives (Table I) are included in the preparations to kill or to inhibit the growth of micro-organisms inadvertently introduced during manufacture or use. They are used in sterile preparations such as eye drops and multidose injections to maintain sterility during use. They may be also added to aqueous injections that cannot be sterilized in their final containers and have to be prepared using aseptic precautions. Preservatives are also used in cosmetics, foods, and non sterile pharmaceutical products such as oral liquids and creams to prevent microbial spoilage. They are not used indiscriminately, and preparations that should not contain preservatives include; injection into cerebrospinal fluids, eye or heart ³ .

The British pharmacopoeia (BP) ⁵ stated that the addition of antimicrobial preservatives to radio-pharmaceutical preparations in multidose containers is not obligatory unless their addition is prescribed in the monograph.

Antimicrobial preservatives are classified into two main sub-groups: anti-fungal preservatives and anti-bacterial preservatives.  Anti-fungal preservatives include compounds such as benzoic and ascorbic acids and their salts, and phenolic compounds such as methyl, ethyl, propyl and butyl p-hydroxybenzoate (parabens).  Antibacterial preservatives include compounds such as quaternary ammonium salts, alcohols, phenols, mercurials and biguanidines.

II. B.  Antioxidants :

Antioxidants (Table II) are included in the pharmaceutical products to prevent deterioration from oxidation. Antioxidants are classified into 3 groups. The first group is known as true antioxidants, or anti-oxygen, probably inhibit oxidation by reacting with free radicals blocking the chain reaction. Examples are alkygallates butylated hydroxyanisol, butylated hydroxytoluene, nordihydroguaiaretic acid and the tocopherols.  The second group consists of reducing agents; these substances have lower redox potentials than the drug or adjuvant which they are intended to protect, and are therefore, more readily oxidized. Reducing agents may act also by reacting with free radicals. Examples are ascorbic acid, the potassium and sodium salts of sulphurous acid. The third group consists of antioxidant synergists which usually have little antioxidant effect themselves but probably enhance the action of antioxidants in the first group by reacting with heavy metal ions which catalyze oxidation. Example of antioxidant synergists are citric acid, editic acid and its salts, lecithin and tartaric acid ^6-8 . The classification of the antimicrobials and antioxidants into groups with typical examples and structural formulae are abridged in Tables (I) and (II), respectively.

Table (I): Classes of Antimicrobial preservatives :

Table (I): Cont.:

Table (II): Classes of Antioxidants

{mospagebreak title=Analysis of Preservatives }

III. Analysis of Preservatives

Single preservative, but more often combinations of preservatives, are commonly used in pharmaceuticals, cosmetics, biological samples, food, wood, and plastics products to prevent alteration and degradation of the product formulations. However these preservatives may be harmful to consumer due to their tendency to induce allergic contact.  Hence the simultaneous determination of these preservatives in commercial pharmaceutical products is particularly important both for quality assurance and consumer safety ⁹ . Therefore, analytical methodologies developed for the quantification of preservatives in these matrices are usually designed to overcome the problems associated with interferences which are originated from other constituents. Because of the circumstances for which they are used in pharmaceutical products, preservatives are usually found as minor components in complex matrices.

There are several reviews for the analysis of preservatives in food ^10,11 , wood ^11,12   polymers ¹³ , biological samples ^14-16 , and cosmetics ^17,18 . However, the literature is poor in terms of a comprehensive review on the analysis of preservatives collectively in pharmaceutical products, except for the review published in Chinese language by Ho and Chen ¹⁹ . The present review is devoted for the analysis of preservatives in pharmaceutical products.

III. A.  Analysis of antimicrobial preservatives:

Antimicrobial preservatives have been analyzed by both microbiological ⁴ and chemical methods.  The scope of this review will outline the chemical procedures.

III. A. 1. Parabens :

Parabens are ester of р-hydroxybezoic acid (Table I), they are included in phramceutical products and cosmetics as antimicrobial preservatives. The methyl and propyle esters are most frequently employed parabens. These materials frequently contain, ethyl paraben, butyl paraben and р-hydroxybenzoic acid as impurities ²⁰ . Methylparaben is the most soluble ester, while the higher members have saturation solubility above the needed concentrations for preservative effect.  Unlike benzoic acid, parabens retain their antimicrobial activity at raised pH values (7-9) ²¹ .

Chemically, parabens are stable in acid medium and even at elevated temperatures, but hydrolysis will occur as the pH increases especially in strong alkaline solutions.  The higher esters exhibit the greatest antimicrobial activity but this is offset by a lower solubility.  To achieve maximum activity, parabens are used as mixtures of esters since they have synergistic effect ⁷ . The European Pharmacopoeia ²² assay for р-hydroxybenzoate preservatives involves saponficatin followed by titration of a sample, and a TLC method for identification.

Chromatographic methods such as high performance liquid chromatography (HPLC), capillary electrophoresis (CE) and UV derivatives spectrophotometry have been reported as selective methods for the determination of parabens in pharmaceutical products. The reported methods of analysis can be classified as follows:

III. A. 1.a. Capillary electrophoresis methods:

Electrokinetic capillary electrophoresis (CE) was reported for the determination of methyl, ethyl, propyl and butyl parabens in cosmetic preparations using fused silica capillary column and applied voltages of 22, 25, 27 and 30 kV respectively. The UV detection was carried out at 220 nm ²³ .  Heo and Lee ²⁴ reported micellar CE for the determination of 4- hydroxybenzoic acid and its methyl, ethyl and propyl esters (parabens) in liquid preparations using fused silica capillary column and an applied voltage of 30 kV with UV detection at 205 nm. Labat et al ²⁵ , reported the use of CE and HPLC methods for the determination of parabens in cosmetic products.  CE was carried out using fused silica gel column and an applied voltage of 30 kV with UV detector at 290 nm.

Micellar Electrokinetic chromatography (MEKC) and microemulsion electro- kinetic chromatography (MEEKC) were reported as analytical methods for the simultaneous separation and determination of preservatives in commercial drug products. The determined preservatives were parabens, benzoic acid, sorbic acid, imidurea, triclosan and dehydroacetic acid. Fused silica capillary column was used with an applied voltage of 25 KV. Detection was carried out at 200 nm ²⁶ . MEKC method was reported by Driouich et al ²⁷ , for the determination of haloperidol tranquilizer, parabens and benzoic acid using fused silica capillary column and an applied voltage of 30 kV with UV detection at 200 nm. Imidurea and parabens, in ointments were determined by MEKC. Fused silica capillary column was used with an applied voltage of 30 kV. Detection was carried out at 200 nm ²⁸ .

MEEKC method was reported by Mahuzeir et al , for the determination of         4- hydroxybenzoic acid and its derivatives parabens using fused silica capillary column and an applied voltage of 11kV with UV detection at 200 nm ²⁹ .

III. A. 1. b. High performance liquid chromatographic methods

High performance liquid chromatographic methods (HPLC) are clearly the most suitable technique if complex samples are involved. So, several HPLC methods were reported for the determination of parabens in pharmaceutical products. Regular or silanol-deactivated C18 or C8 columns coupled with UV detection are the most common configuration for the analysis of parabens in pharmaceutical products and cosmetics. This review refers only to the most recent HPLC methods for the determination of parabens in various pharmaceutical products.

Parabens in liquid pharmaceutical products were determined by RP-HPLC using C18 column. The mobile phase was a mixture of methanol-and phosphate buffer of pH 7.05. Detection was carried out at 254 nm ³⁰ . RP-HPLC with C18 column was also reported for the determination of methyl and propyl parabens besides hydrocortisone and its degradation products in a topical cream ³¹ . The mobile phase was a mixture of methanol-acetontrile and water. Detection was carried out at 238 nm. A similar configuration, with a different pH mobile phases (pH 2.5), was used for the analysis of parabens with diclofenac sodium and its degradation products ³² . Another RP-HPLC configuration, with a different mobile phase (at pH 3.45 with glacial acetic acid), was used for the analysis of parabens with ambroxol expectorant. Detection was carried out using at 247 nm ³³ .

RP-HPLC with C8 column was reported for the determination of propyl parben, butyl parben, cisapride and its oxidation products, using acetonitrile phosphate buffer at pH 7 as a mobile phase. Detection was carried out at 276 nm ³⁴ . Also, RP-HPLC with C8 column, with a different pH mobile phase of pH 4 was used for the analysis of parabens in pediatric suspension containing indinavir (anti-HIV type A virus), and potassium sorbate. Detection was carried out at 260 nm ³⁵ . Similar procedures were developed for the separation of parabens from metronidazole benzoate ³⁶ ; for the determination of parabens and chlorpromazine in oral solutions ³⁷ ; for the determination of parabens in cough-cold syrup containing bromhexine HCl, and dextromethorphan HBr ³⁸ ; or for the determination of parabens in presence of phenoxetrol and croconazole HCl in aqueous solutions ³⁹ . The system described by Kollmorgen and Kraut ³⁷ was also used to study the stability of parabens.

Mangani ⁴⁰ developed an HPLC method for the determination of parabens and sorbic acid in cream or cough syrup where an on-line clean up trace-enrichment cartridge (TCE) was introduced.  In this system, the analytes was transferred to the TCE for sample clean up via the mobile phase and slowly eluted onto the C8 column whilst any interferents were retained on the TCE.  Following clean up, the TCE was regenerated by back-flushing with acetonitrile. UV detector was used.

Similar procedures were reported for the analysis of parabens in radiolabeled pharmaceuticals containing Tc 99-M mebrofenin ⁴¹ for the simultaneous quantization of parabens and ketoprofen in commercial gel formulations ⁴² ; for the analysis of parabens in dosage forms ⁴³ and also for the separation and analysis of parabens and desonide after solid-phase extraction on Bond Elute cartridges packed with silica, diol or aminopropyl sorbents ⁴⁴ .

Smith and Burgess ⁴⁵ used superheated H ₂ O as an environmentally clean and safe mobile phase for the HPLC analysis of mixture of parabens or phenols without an organic solvent modifier.  A C18 column operated at 120 ⁰ C was used for phenols and at 170 ⁰ C for parabens.  Detection was carried out at 254 nm.

Methyl paraben in preparations containing methadone was determined by HPLC using cyano-column and detection at 259 nm ⁴⁶ . Other HPLC detectors have been also used in the analysis of parabens in pharmaceutical products.  For example, RP-HPLC system coupled with amperometric detection at a glassy carbon electrode at +1.25 V vs. Ag/AgCl was used for the determination of parabens and thiomersal in aqueous solutions or in pharmaceutical preparations ⁴⁷ . Garcia-Sanchez et al ⁴⁸ , utilized alpha-cyclodextrin as a restricted access mobile phase for RP-HPLC with fluorimetric detection of 4-hydroxybenzoic acid and methyl paraben.

RP-HPLC was also utilized in determining the degradation products of parabens.  For examples, Dhaliwal and Theobald ⁴⁹ utilized a C18 column and UV detection for the analysis of parabens and their degradation products after liquid-liquid extraction into CH ₂ Cl ₂ from an acid medium. Thompson et al ^50,51 , investigated the principal factors responsible for the appearance of unknown peaks that had arisen from the degradation involving interaction between parabens and sorbitol and/or glycerol (polyols).  Isocratic and gradient elution techniques were used.

Physicochemical screening of antimicrobials including parabens, BKCs and alcohol as potential preservatives for submicron emulsion was carried out by Sznitowska et al ⁵² . The physicochemical studies were carried out utilizing HPLC with C8 column. Detection was carried out at 254 nm. Other HPLC procedures were also described for the determination of parabens in pharmaceutical products ⁵³ .

III. A. 1. c. Gas chromatographic methods:

The US Pharmacopoeia ⁴ recommends gas chromatography (GC) for the determination of parabens as preservatives using FID.  De-Croo et al ⁵⁴ , used GC for the determination of parabens in antacid products.  Parabens were extracted, and then derivatized by reaction with hexamethyldisilazane and heptafluorobutyric anhydride.  Several methods were reported for the determination of parabens and their derivatives using the same technique with different derivatizing agents ^55-58

III. A. 1. d.  Thin layer chromatographic methods :

Several Thin layer chromatography (TLC) and HP-TLC procedures have been reported for the determination of parabens in pharmaceutical products. European Pharmacopoeia ²² specifies a TLC method for the detection of parabens in pharmaceutical compounds.

Parabens in antibiotic peptides; neomycin sulphate, polymixin B sulphate and zinc bacytracin ointments were determined by TLC, using n-pentane-glacial acetic acid mixture as a mobile phase. Detection was carried out by densitometry measurement at 260 nm ⁵⁹ . Similarly, parabens in antacid suspension were determined by RP-TLC plates. Detection was carried out by densitometry measurement at 254 nm ⁶⁰ . Similar procedure was developed by Thomassin et al ⁶¹ , where a comparative study of the application of HP-TLC and HPLC in the analysis of parabens was initiated.  For HP-TLC, fluorescent RP-TLC plates were used and the detection was at 260 nm. For HPLC, C18 column was used with aqueous methanol as a mobile phase and UV detection at 254 nm.

The two dimensional, tw o- phase TLC techniques were also applied for the determination of parabens and carboxylic acids in pharmaceutical products ⁶² . Sample was applied to the bottom left corner of a silica gel plate and then developed by normal-phase.  The plate was dried and was then developed from the right-edge with another mobile phase. Visualization was under UV light; parabens were separated as a single spot whilst the carboxylic acids moved with the solvent front. The method was applied to two groups to illustrate the applicability of the technique.

Tomankova and Pinkasova ^{63, 64} reported the use of HPTLC for the quantification and the kinetic study of degradation products of parabens in pharmaceutical products, such as suspensions and ointments.

III. A. 1 .e. Flow injection analysis:

 Recently, a flow injection–chemiluminescence (FIA-CL) method has been reported for the determination of methyl, ethyl, propyl and butyl parabens in food, pharmaceutical products and cosmetics ⁶⁵ . The method is based on the fact that parabens greatly enhance the chemiluminescence reaction between the cerium (iv)-rhodamine system in strong sulphuric acid. The method has very low detection limits and wide dynamic range.

III. A. 1.  f. Spectrophotometric methods :

UV‑visible spectrophotometry is still considered to be a convenient and low cost method for the determination of preservatives. Several spectrophotometric and colorimetric methods have been reported for the determination of parabens in bulk material. While in pharmaceutical products, the presences of active ingredients, other preservatives and excipients prevent a direct conventional spectrophotometric analysis, due to severe spectral overlap. To overcome such an overlap, a second order derivative spectrophotomeric method was developed by Popovic et al for the determination of methyl parabens, propyl parabens and bifonazole in cream ⁶⁶ . The method is based on measurements of acidic extract at 241.5 nm.

Partial least-squares calibration method for the UV-spectrophotometric determination of parabens and ketoprofen in a gel preparation was developed by Blanco et al ^67. In that method, the sample was extracted into methanol, and NaOH solution was added before measurement at 240-330 nm.

Ouanes et al ⁶⁸ , described an UV-derivative spectroscopic method for the analysis of parabens and haloperidol in preparations. First-derivative spectroscopic method was also applied for the determination of parabens, benzyl alcohol and phenol in different pharmaceutical products ^{69, 70} . The measurements were made at 240-286 nm.

III. A. 2. Acids and Salts

Benzoic acid, sorbic acid and their potassium salts are widely used as food, cosmetics and pharmaceutical preservatives. They exhibit inhibitory activity against a wide variety of fungi, yeast, molds and bacteria, including food born pathogens.        A broader spectrum of microbecidal activity is often achieved by using combination of them, which inhibits several bacterial strains better than either alone ⁷¹ . The acceptable body intake determined by joint FAO/WHO for benzoic acid and sorbic acid are within the range of 0 to 5 and 0 to 25 mg /Kg body mass respectively ⁷² .

Benzoic acid has pKa of 4.2 and sorbic acid has pKa of 4.8 and only the acid forms possess antimicrobial activity. Therefore, their activities are greatest at acid pH values.  The use of these preservatives is restricted to products with a pH value less than 5.  Benzoic acid is chemically stable, but sorbic acid is sensitive to light and air and may require the addition of an antioxidant or refrigerated storage to increase its stability ²¹ .

Several assay methods have been reported for the analysis of benzoic acid, sorbic acid and their salts, in pharmaceutical products and biological materials. Some of these methods were discussed under parabens. The reported methods of analysis of sorbic and benzoic acids and their salts can be classified as follows:

III. A. 2. a. Capillary electrophoresis methods:

Micellar Electrokinetic chromatography ^26,27 and microemulsion electrokinetic chromatography ²⁶ were used for the determination of benzoic acid, sorbic and parabens in pharmaceutical products. These methods were discussed under parabens.

III. A. 2. b. High performance liquid chromatographic methods:

In addition to the methods mentioned under parabens, the following HPLC methods have been reported for the analysis of sorbic and benzoic acids and their salts. Mikami et al ⁷³ , developed solid phase extraction and HPLC method for the simultaneous determination of dehydroacetic acid, benzoic acid, sorbic acid and salicylic acid in cosmetics, using tetra-n-butyl ammonium hydroxide as an ion pair reagent. El-Gindy et al ⁷⁴ , developed RP-HPLC, in addition to two spectrophotometric methods;  principle component regression and partial least squares methods for the determination of sorbic acid, cyproheptadine and soluble multivitamins. HPLC analysis was achieved at 220 nm and 288 nm. Bousquet et al ⁷⁵ , reported a pre-column derivatization with 1(2,5-dihydroxyphenyl)-2-bromoethanone, followed by RP-HPLC method for the  determination of sorbic acid in cosmetics. Detection of the derivative was carried out electrochemically at potential of +0. 45 V.

RP-HPLC with C8 column using an UV detector at 247 nm method was described for the determination of ambroxol HCl and benzoic acid in syrup ⁷⁶ .

Sorbic acid was determined by RP-HPLC with C18 column in creams ⁷⁷ . Stability studies of sorbic acid and bromhexine in cough syrup was carried out by RP-HPLC using C18 column ⁷⁸ . The results of the study indicated that, no decomposition was observed for both compounds after 26 months of storage at room temperature.  Stability studies of sorbic acid and other preservatives using HPLC and spectrophotometry were reported by Mc-Carthy ⁷⁹ . The results of his study indicated that HPLC method is reliable for the determination of the potency of preservatives in presence of their degradation products, while, spectrophotometric method is not.

Ion chromatographic technique was also applied for the analysis of these acids in pharmaceutical products. For example, ion chromatography with conductomeric detection using cation exchange resin column was recently applied for the determination of benzoic acid and some carboxylic acid in water ⁸⁰ . Chen and wang ⁸¹ , reported the usage of ion chromatography with UV detector for the determination benzoic acid and sorbic acid in pharmaceutical products.  Paciolla et al ⁸² , reported ion chromatographic method with UV detector for the determination benzoic acid, sorbic acid and amino-compounds, (like theophylline, pseudo-ephedrine, diphenhydramine) in cough products. Also, benzoic and sorbic acids were determined by HPLC-ion chromatography with conductmetric detection ⁸³ .

III. A. 2. c. Thin layer chromatographic methods:

The two dimensional ⁶³ , tw o- phase TLC technique was discussed under parabens and its application for carboxylic acids in pharmaceutical products.  El-Bayoumi et al ⁸⁴ , developed a TLC method for the determination of parabens, benzoic and sorbic acids in bulk powder, and pharmaceutical formulations using different developing solvents and UV detection at 254 nm.

Mandrou and Bresolle ⁸⁵ reported another TLC method for the determination of sorbic acid and benzoic acid in pharmaceutical products and biological fluids                       

III. A. 2. d. Spectrophotometric methods:

The principal-component regression method and first derivative spectrophotometric method was developed for the analysis of benzoic acid in the presence of cinitapride in syrup ⁸⁶ . In addition, benzoic acid and parabens were determined in pharmaceutical products (like, cellulose gel, KI solution, and glycerin and stearine ointments) by direct UV spectrophotometry at 256 nm in 0.1 M HCl.  Interference by KI was avoided by measurment in 0.1 M NaOH at 296 nm.  Sorbic acid in syrups was determined at 250 nm ⁸⁷ .

III. A. 2. e. Electrochemical methods:

Sorbic acid and parabens were determined by coulometric technique following the procedure described by Nikolic et al ⁸⁸ .

III. A. 3. Quaternary Ammonium Compounds:

Quaternary ammonium compounds have been in clinical as antimicrobial additives use since 1935. They have been used to maintain sterility of a variety of prescriptions and OTC products such as cosmetics, infant care products; pharmaceutical nasal sprays ophthalmic solutions and otic drops ⁸⁹ . Marpel et al ⁹⁰ prepared a review article about the safety of benzalkonium chloride in intranasal solutions .

Several chemical derivatives of the quaternary ammonium ion have been used as preservatives. The most frequently used individuals are benzalkonium chlorides (BKCs), cetylpyridinium bromide (CPB), cetylpyridinium chloride (CPC) cetrimide, and benzaethonium chloride (BZC) (Table I).

BKCs are a mixture of alkylbenzyldimethyl ammonium chlorides with the general formula [C ₆ H ₅ CH ₂ N (CH ₃ ) ₂ R] ⁺ Cl ^- , where R = n-C ₈ H ₁₇ to n-C ₁₉ H ₃₉ . The n-C ₈ H _17, n-C ₁₂ H ₂₅ and n-C ₁₆ H ₃₃ homologues comprise the major portions of the alkyl mixture.  The C12 homologue is most effective against moulds, yeast and fungi, the C14 homologue is most effective against gram-positive bacteria, and the C16 homologue is most effective against gra m- negative bacteria ⁹¹ . The ratio of alkyl homologues must meet specific USP ⁴ requirements. These requirements state that the C12 homologue must comprise at least 40% of the total BKCs content, and that the C14 homologue must be at least 20%.  Furthermore, these two homologues together must comprise at least 70% of the total content.  Cetrimide is the tetra-decyl tri-methyl ammonium bromide with a small amount of dodecyl and hexadecyl derivatives ⁹² . 

Quaternary ammonium salts are chemically stable in aqueous solutions, will withstand autoclaving, and compatible with heavy metals, alkalis, oxidants and anionic surfactants ²¹ .  The reported methods of analysis can be classified as follows:

III. A. 3. a.  Capillary electrophoresis methods:

The developed analytical procedure demonstrates that CE is a reliable and sensitive method, and offers a convenient analytical technique to determine quaternary ammonium compounds, due to the presence of a positive charge. For these reasons, several analytical methods based on utilization of capillary electrophoresis (CE) technique have been reported for the determination of this group of preservatives.  Hou et al ⁹³ , developed a capillary zone electrophoresis (CZE) method to determine BKCs in ophthalmic solutions; the sample was separated at a potential of 15 kV and detected with UV-VIS detector at 200 nm. CZE was superior over HPLC in terms of sensitivity and precision.

Mixtures of cationic surfactants; BKCs and CPC were resolved and determined using CZE and MEKC-CZE in industrial house hold surfactants.  The MECK method has an excellent resolution for all house hold solutes including C12-C18 homologues ⁹⁴ .  Prince et al ⁹⁵ , utilized HPCE for the separation and determination of BKCs and their homologues on silica capillary column and UV detection at 214 nm.  However, HPCE was more sensitive and precise compared to the HPLC. Also, BKCs in nasal drops containing naphazoline, dexamethasone were determined by a similar procedure developed by Raith et al ⁹⁶ .

Comparative studies for the determination of BKCs utilizing HPLC coupled with UV detection at 250 nm, CE coupled with UV detection at 254 and 214 nm, and first derivative spectroscopy was developed by Bernal et al ⁹⁷ .  These methods were successfully applied for the determination of BKCs in the presence of beclomethasone dipropionate (or fluticasone propionate) active ingredients and excipients in nasal sprays.

BKCs, 2-phenylethanol and beclomethasone dipropionate in nasal spray products were separated and determined with HP-CE.  Fused-silica tubes were used at 20 ⁰ C and 50 ⁰ C with UV detection at 214 and 254 nm, respectively ⁹⁸ .

Jimidar et al ⁹⁹ , utilized CE coupled with UV detection at 215 nm for the determination of BKCs in pharmaceutical products. C12 and C14 -BKCS derivatives were separated and quantified. BKCs were also separated from histamine acid phosphate and determined using imidazole as an internal standard by CE where the separation was conducted at 30 ⁰ C with detection at 200 nm ¹⁰⁰ .

Cetylpyridinium chloride in mouth wash was determined by CE with indirect UV detection or by HPLC with conductometric detection ¹⁰¹ .

III. A. 3. b. High performance liquid chromatographic methods:

HPLC methods with different impacts of column packing type, mobile phase composition, buffer, ionic strength and column temperature, were described for the determination of quaternary ammonium compounds in various pharmaceutical products. Amino functional group ¹⁰² , reversed phase ^103,104 and cyano ^105-106 packing columns were developed. The European Pharmacopoeia ²² specifies HPLC for the determination of BKCs in pure form. In addition to the methods mentioned before, the following methods have been reported for their determination in pharmaceutical products. BKCs, in aerosol preparations, were determined by HPLC with cyano-column and the mobile phase was aqueous acetonitrile at pH 5, and the detection was performed at 262 nm ¹⁰³ .

Bernal et al ¹⁰⁴ , utilized the HPLC with reverse phase column and acetonitrile-water mixture as a mobile phase for the determination of BKCs in pharmaceutical products. The detection was performed at 210 nm. In ophthalmic solutions, Fan and Wall ¹⁰⁵ and Kummerer et al ¹⁰⁶ , determined BKCs by HPLC with cyano column after the sample has been extracted to solid phase. Ambrus et al ¹⁰⁷ , described a direct RP-HPLC method with UV-VIS detector for the determination of BKCs in ophthalmic systems.

BKCs and their homologues were separated and determined by HPLC using cyano-column and UV detection at 254 nm. BKCs and nonoxynol-9 in preparations were determined by RP-HPLC during a pre-formulation study on cyano column and UV detection at 214 nm ¹⁰⁸ .

Parhizkari et al ^{109, 110} , developed two stability indicating ion-pair RP-HPLC methods for the determination of BKCs in ophthalmic solutions where C12- and C14-homologues were separated and quantified.  Also, BKCs were determined in eye care products by HPLC; they were separated from various products and concentrated by either solid-phase extraction onto C18 cartridges or by an online column-switching technique ¹¹¹ . Other HPLC procedures with UV detection were reported for the analysis of quaternaries include determination of BKCs in ophthalmic solutions using phenyl column ¹¹² and cyano-column ¹¹³ in addition to the determination of CPC in pharmaceutical and cosmetic products ¹¹⁴ .

III. A. 3. c. Gas chromatographic methods:

This class of compounds is not amenable to direct GC analysis due to volatility problems.  However, the volatility is greatly enhanced by pyrolysis to tertiary amines and subsequent GC. This procedure was described by Suzuki et al ¹¹⁵ . In that procedure, BKCs were reacted with potassium t-butoxide, and extracted into benzene. The extract was analyzed on SE-30 column and the produced alkyl homologues were quantified. These procedures were similar to the procedure developed by Cybulski ¹¹⁶ where, BKCs were converted to two tertiary amines: alkyldimethyl amine and alkylbenzylmethyl amine. The pyrolysis products of BKCs were identified by GC-MS.

III. A. 3 .d. Thin layer chromatographic methods:

The chain homologues of BKCs, cetrimide, CPB and CPC were separated on TLC and detected by the yellowish-brown spots on a colorless background. They were quantified by UV densitometry ^117,118 .

III. A. 3. e. Flow injection analysis:

Owing to the presence of positive charges, quaternary ammonium compounds have higher ability towards ion-pair reactions with acidic dyes. The formed colored ion pair products were extracted into a suitable organic solvent and measured by the spectrophotometric technique. Sakai ¹¹⁹ reported a FIA procedure for the analysis of BKCs after ion-pair formation with tetrabromophenolphthalein ethyl ester reagent in CH ₂ Cl ₂ followed by phase separation and detection at 610 nm with the flow-cell operated at 45 ⁰ C.  Halvax et al ¹²⁰ _, developed a FIA method for the determination of BKCs in pharmaceutical products containing xylometazoline, timolol, phenylephrine or carbachol by on-line ion-pair extraction of BKCs with picrate into CHCl ₃ and detection at 370 nm.

The determination of BKCs after the reaction with quinine - bromophenol blue present in the carrier stream, followed by on-line extraction and measurement at 610 nm. This method was reported by Miyaji et al ¹²¹ .

BZC in pharmaceutical products was determined by FIA where the sample was injected in stream containing quinidine and bromochlorophenol blue in 1,2-dichloroethane. The product in the organic layer was measured at 605 nm ¹²² .

The interference of thiomersal and BKCs in the simultaneous determination of phenylephrine HCl and pheniramine maleate products was eliminated by passage through beds of anion-exchange resin and then through cation-exchange resin, using two porous-membrane phase separators and determined by FIA with photometric detector ¹²³ .

III. A. 3. f. Spectrophotometric methods:

Several spectrophotometric procedures have been reported in the literature for the determination of quaternary ammonium compounds in pharmaceutical products. Gorog ¹²⁴ , in his text book reviewed spectrphotmetric method for the determination of quaternary ammonium preservatives.

Preservatives including cetyltrimethyl ammonium bromide, CPB, and dimethyldodecylbenzyl ammonium bromide react with 1-(4-nitrophenyl)-3-(4-phenylazophenyl) triazene and the produced colors are measured at 530, 540 and 590 nm, respectively ¹²⁵ .

A colorimetric method for the determination of BKC in eye-drops based on ion-pair formation between BKC and eosin was developed by Hadady and Fabian ¹²⁶ . The method is based on measurement of the decrease in absorbance of eosin ^. A colorimetric method based upon ion pair association between quaternary ammonium salts, such as CPC, and tetraiodofluorescein and quinine at pH 7 in the presence of 1,2-dichloroethane was developed by Sakai et al ¹²⁷ .

Two spectrophotometric methods were developed by Belal et al , for the determination of BKC in ophthalmic preparations based on the formation of highly colored charge-transfer complexes with 7,7,8,8-tetracyanoquinodimethane and 2,3-dichloro - 5,6-dicyano -p- benzo-quinone  BKC in pharmaceutical products ¹²⁸ .

Colorimetric procedures for the determination of BKCs were developed through reaction with bromophenol blue followed by organic solvent extraction and measurement at 610 nm ¹²⁹ or reaction with 4-(4-nitrophenylazo) resorcinol and NaOH and measuring the absorbance at 630nm. ¹³⁰ . Also, a direct UV measurement of this group of compounds was also reported ^131-133 .

III. A. 3. g. Titrimetric methods :

Titrimetric methods were also reported for the determination of quaternary ammonium compounds. These methods are based on coupling of the positive charge with negatively charged titrants. End points were detected potentiometrically or using visual indicator.

BZC in pharmaceutical preparations was determined by titration with sodium tetraphenylborate or sodium dodecyl sulfate and the end point was detected by potentiometer using ISE ¹³⁴ . A similar procedure was reported by Satake et al ¹³⁵ , where BKCs and zephiramine in a bactericidal solution were determined by titration with sodium tetraphenylborate using ISE sensitive to tetraphenylborate.

CPC, BKCs, acrinol (ethacridine lactate), and methylephedrine were determined, using sodium tetraphenylborate as titrant and tetrabromophenol-phthalein ethyl ester as indicator ¹³⁶ . Other titrimetric procedures for the determination of quaternary ammonium compounds are also available ^137-140 .

III. A. 4. Mercurial Compounds:

Mercurial compounds have been used as preservatives in pharmaceutical products for many years. Although, their antimicrobial activities are not affected by pH, and temperature, their uses were declining, due to their biological toxicity and environmental pollution ²¹ . Moreover, they are not compatible with sulfides, thiol-containing compounds, halides, aluminum and other heavy metals. Mercurial compounds are not recommended in situations where prolonged administration is necessary. 

Limited methods for the analysis of this group of compounds in pharmaceutical products have been reported in the last decade. The commonly used members of this series are, phenyl mercury salts [acetate (PMA), borate (PMB) or nitrate (PMN)] and thiomersal (Table 1).  The reported methods of analysis devoted to mercury salts can be classified as follows:

III. A. 4. a. High performance liquid chromatographic methods:

Several HPLC methods have been utilized for the determination of mercurial compounds in pharmaceutical preparations.  In addition to the methods mentioned before, the following procedures have been reported:-

Larroque and Vian ¹⁴¹ reported RP-HPLC with C18 column for the determination of phenylmercuric nitrate in pharmaceutical products. The detection was carried out using UV detector at 258 nm.  The mobile phase was water, acetonitrile, and EDTA, mixture.

Thiomersal in ophthalmic products was determined by RP HPLC with C18 column. The detection was affected using inductively coupled plasma mass-spectrometric detector. The mobile phase was buffered-water, methanol and acetonitrile mixture ¹⁴² .

RP ion-suppressed chromatography, RP ion-pair chromatography and ion chromatography in combination with amperometric and coulometric detectors were reported for the determination of thiomersal and its degradation products;                  2-mercaptobenzoic acid and 2,2'-dithiodibenzoic acid. The detection was carried out using electrochemical detector at carbon electrode ¹⁴³ . Also, thiomersal and its decomposition products after exposure to radiation from artificial UV light were analyzed on a RP-HPLC with C18 column with buffered water / methanol as mobile phase and electrochemical detection at 1.2 V vs. Ag/AgCl electrode ¹⁴⁴ .

III. A. 4. b.  Spectrophotometric methods:

 A spectrophotometeric method for the determination of phenylmercuric nitrate in ophthalmic ointments and eye drops was reported by Anjaneyulu et al ¹⁴⁵ . The method is based on the formation of a ternary complex between Hg (II) (produced after digestion by HCl-HNO ₃ ) with 4-(2-pyridylazo) resorcinol and benzyl-hexadecyldimethyl ammonium chloride in phosphate buffer of pH 7.2. The reaction product was extracted into CHCl ₃ and measured at 525 nm.

III. A. 4. c.  Atomic absorption spectroscopic methods:

Thiomersal and phenylmercuric bromide in pharmaceutical products were determined by AAS after digestion with a mixture of HCl and HNO ₃ . Hg was measured by direct aspiration into an air-acetylene flame; in this case, 0.2% of K ₂ Cr ₂ O ₇ solution was added to obtain a stable baseline.  Also, Hg was determined by use of the flameless technique with a mercury hollow-cathode lamp ¹⁴⁶ . Meakin and Khammas ¹⁴⁷ determined thiomersal at preservative concentration by flameless AAS.

III. A. 4. d.  Electrochemical methods:

Polarographic methods were reported for the determination of thiomersal and phenylmercuric nitrate ^{4, 148} .

Pilar-da-Silva et al ¹⁴⁹ , developed an amperometric method for the determination of thiomersal in soft contact lens solutions, where the cyclic voltammogram was recorded from -0.4 to +1 V. A voltammetric procedure for the determination of PMB in pharmaceutical products based on the use of a carbon paste electrode modified with amberlite was reported by Cai t al ¹⁵⁰ .

Bertocchi et al ¹⁵¹ , reported an enzymatic amperometric procedure for the determination of thimersol in ophthalmic products. The method is based on the inhibition effect of mercury on invertase and a sucrose electrode.

III. A. 4. e.  Titrimetric methods:

Nagy and Szasz ¹⁵² described a direct potentiometric method for the determination of PMB with sodium tetraphenyl borate using ion-selective electrode (ISE). Another titrimetric procedure was reported by Wood and Welles ¹⁵³ for the determination of PMN in aqueous solutions. The method was based on the formation of insoluble phenylmercuric iodide upon titration with potassium iodide, and the end point was detected with iodide-iodide selective electrode.

III. A. 5. Alcohols:

There are many alcohols that possess antimicrobial activity, a few of them are used as preservatives; such as arylalkyl and highly substituted aliphatic alcohols (Table I).  Alcohols are usually used in combination with other preservatives; for example phenoxyethanol with parabens and phenylethanol with BKCs.  Both compounds are thermally stable but phenylethanol is susceptible to oxidation ²¹ . Benzyl alcohol in pharmaceutical products is often used as a solvent and a preservative, it is slowly oxidized to benzaldehyde and benzoic acid, so, the USP ⁴ limits the presence of benzaldehyde in benzyl alcohol to the level of 0.2% with the quantification by HPLC.  The British Pharmacopoeia ⁵ stated that, benzyl alcohol used for parentral dosage form should not contain more than 0.05 % of benzaldehyde and described a GC method for its determination in raw materials.

Highly substituted aliphatic alcohols such as chlorbutanol are stable in acid medium but unstable at high temperatures or in alkaline medium. Bronopol              (2-bromo-2-nitro - propan-1,3-diol) is a new member of this group and is widely used in both pharmaceutical and cosmetic products due to high activity and low toxicity ²¹ . Ethanol is not normally used as a preservative due to the high concentration needed ^.21 . The reported methods of analysis of alcohols can be classified as follows:

III. A. 5. a. High performance liquid chromatographic methods:

 The literature is enriched with HPLC methods for the determination of alcoholic preservatives in pharmaceutical products. Some of these methods were discussed before.

RP-HPLC procedures with different columns and UV detection modes were reported for the determination of benzyl alcohol in injections ⁵⁴ , in cream containing chlotrimazole and mometasone furoate ¹⁵⁵ and in topical preparations ¹⁵⁶ . Benzyl alcohol and its oxidative product, benzaldehyde, were determined in parentral solutions by RP-HPLC using C8 column and UV detection at 240 nm ^{157, 158} . Pre-column derivatization HPLC methods were developed for the determination of benzyl alcohol in different pharmaceutical products ^{159, 160} .  HPLC with a fluorescence detector was utilized for the determination of benzyl alcohol and other alcohols after derivatization with 3,4-dihydr o- 6,7-dimethoxy-4-methyl-3-oxoquinoxaline-2-carbox-azide in benzene ¹⁶¹ . Other HPLC methods for the analysis of benzyl alcohol in different dosage products were also reported ^{162, 166} .

Ion-pair HPLC ¹⁶⁷ procedure was reported for the kinetic determination of the degradation products of bronopol in pharmaceutical and cosmetic products.            RP- HPLC procedure was described by Ferioli et al , for the analysis of bronopol in pharmaceutical preparations ¹⁶⁸ .

III. A. 5. b. Gas chromatographic methods :

The USP ⁴ , specifies a GC method using FID for the determination of chlorbutanol and benzyl alcohol. A capillary GC equipped with FID and fused silica column was reported for the determination of benzaldehyde arising from benzyl alcohol in injectable products ¹⁶⁹ . Also, benzaldehyde, as an impurity in benzyl alcohol, was determined by GC and difference-spectrophotometric techniques ¹⁷⁰ . Benzyl alcohol in heparin solution for injection was determined by GC using FID ¹⁷¹ .

A capillary GC method coupled with ECD was developed for the determination of chlorbutanol in pharmaceutical products ¹⁷² .

III. A. 5. c. Spectrophotometric methods :

Benzyl alcohol and diclofenac sodium in pharmaceutical products were determined by first and second order derivative spectrophotometry ¹⁷³ .

Benzaldehyde in benzyl alcohol was determinaed by measuring the difference in absorbance at 248 nm, between two equimolar solutions of benzaldehyde; one of them contains sodium bisulfite. The difference in absorbance was characteristic for benzaldehyde, benzyl alcohol did not interfere ¹⁷⁴ .

Sanyal et al ¹⁷⁵ , utilized UV spectrophotometry for determination of bronopol in raw material and in its degradation products.

III. A. 5. d. Electrochemical methods:

An amperometric enzyme electrode was developed for the determination of alcohols. The method is based on immobilizing alcohol oxidase in polynylferrocenium matrix coated on a Pt electrode. The amperometric response was measured at +0.7 V versus SCE ¹⁷⁶ .

The polarographic technique applied for the determination of benzaldehyde arising from the oxidaton of benzyl alcohol in diclofenac sodium products ¹⁷⁷ .  Benzyl alcohol in injection products was determined by polarographic catalytic wave. The method is based on the oxidation of benzyl alcohol into benzaldehyde using peroxydisulphate. The produced catalytic wave was linear to benzyl alcohol concentartion ¹⁷⁸ . 

III. A.  6.  Phenolic Antimicrobials:

Phenol and its derivatives such as o- , p- and m- cresol and chlorocresol Tale II)  are used commercially as disinfectant and still used as preservatives in pharmaceutical products; due to their antimicrobial activities especially below pH 9 ²¹ .

Phenol was the first antimicrobial agent, and is still in use along with other phenolic derivatives. Cresol is a mixture of ortho, para and meta- forms . The meta form is predominating. Chlorocresol is mainly 4-chloro-3-methylphenol. It is difficult to discriminate between the methods of analysis of phenols as disinfectant or as preservatives these methods can be classified as follows:

III. A. 6. a. High performance liquid chromatographic methods:

 Several HPLC procedures have been reported for the determination of phenolic compounds. RP-HPLC methods were reported for the analysis of phenol, thymol, chlorocresol, and chloroxylenol in commercial pharmaceutical products. The method is based on the use of 1-fluoro-2,4-dinitrobenzene as UV labeling reagent in pre-column derivatization ¹⁷⁹ . RP-HPLC coupled with C18 or cyano column method was developed for the determination of chlorocresol in combination with betamethazone using online post-column photochemical derivatization. The chromatogram was irradiated with post-column UV at 254 nm and detected with diode-array at 230-240 nm ¹⁸⁰ . RP-HPLC methods coupled with C18 column and UV detection at 271 nm was reported for the analysis of phenol and cresol in anti-fungal lotions using nicotinic acid as an internal standard ¹⁸¹ .

 HPLC with fluorescence detector has been utilized for the determination of chlorocresol and chloroxylenol in pharmaceuticals ¹⁸² . Other several HPLC methods for the determination of phenol in pharmaceutical products containing atropine ¹⁸³ , phenol and cresol in insulin ¹⁸⁴ , phenol and resorcinol in ointment ¹⁸⁵ and cresol-containing solutions ¹⁸⁶ . Chlorocresol and morphine sulfate in injectable products ¹⁸⁷ were also reported.

III. A. 6. b. Gas chromatographic methods:

The USP ⁴ specifies GC with FID for the determination of phenol as                      a preservative. Solid phase GC-MS method was reported by Kojima et al ¹⁸⁸ . The method is based on the derivatization of phenols with pentafluoropyridine, followed by adsorption of the derivatives on anion-exchange solid phase cartridge. The derivatives were determined by GC-MS. A capillary GC method was developed for the simultaneous analysis of camphor and m- cresol in commercial cream products ¹⁸⁹ .

Capillary GC with FID was reported for the determination of phenol and glutaraldehyde in germicide products ¹⁹⁰ . The steam-carrier gas chromatography technique using packed column and FID was reported for the determination of phenol and salicylic acid in pharmaceutical products ¹⁹¹ . Other methods were established for the determination of phenol, o- , p- and m- cresol and xylenols in pharmaceutical preparations ^192-194 , and chlorocresol in clofibrate ¹⁹⁵ .

III. A. 6.  c. flow injection analysis:

Flow injection and liquid chromatography with on–line quinine sensitized photo-oxidation technique with quenched luminol chemiluminescence (CL) detector were reported for the determination of phenols. The quenched CL for phenols are comparable to those for UV detection at 254 nm ¹⁹⁶ .

A reverse prevaporation FI method coupled with on-line derivatisation and amperometric detection at +0.62 V versus Ag/AgCl, was developed for the determination of trace phenol in aqueous solution ¹⁹⁷ .

A tyrosinase biosensor, constructed by immobilization of the enzyme by cross-linking atop a 3-mercaptopropionic acid, self assembled monolayer on a gold disc electrode was reported for the amperometric detection under FI conditions for several phenolic compounds ¹⁹⁸ .

Chlorocresol in a parenteral pharmaceutical formulation was determined by FIA based on the Liebermann spot test for phenol. The sample was merged with  a reagent stream of nitrous acid and passed through a reaction coil before spectrophotometric detection ¹⁹⁹ .

III. A. 6. d. Spectrophotometric methods:

Wahbi et al ²⁰⁰ , determined phenol in chloraseptic spray spectrophotometrically adopting first-derivative technique. They used the ratios of first-derivative maxima, which are independent of concentration, and the compensated derivative curves. First-derivative synchronous fluorescence spectroscopy technique was also applied for the determination of phenol and resorcinol in pharmaceutical products ²⁰¹ . The diode-array UV/visible spectrometric technique was described for the analysis of phenol and ranitidine by Bourne and Burgess ²⁰² .

Phenol in ear drops was determined by direct measurements of the absorbance in NaOH solution at 288 nm. The calibration graph is rectilinear for 5 to 40 mg/ml ²⁰³ . Phenol and its derivatives in pharmaceutical products were determined through their inhibitory effect on the photolysis of phloxin–EDTA ²⁰⁴ . Samples were added to phloxin - EDTA and borate buffer, then irradiated with a halogen lamp. The time taken for the absorbance of the solution to decrease by 10% was then measured and correlated to phenol concentration.

Phenol and salicylic acid in formulations were determined after azo - dye formation with diazotized sulphanilic acid as a reagent ²⁰⁵ .  Rao and Rao ²⁰⁶ , developed a colorimetric method for the determination of phenol in injections depending on the color reaction with 4-aminoantipyrine. 

III. A. 6. e. Electrochemical methods:

Sotomayor et al ²⁰⁷ developed an amperometric sensor for the determination of  phenolic compounds using a glassy carbon electrode doped with copper dipyridyl  complex as biomimetic catalyst and measurements were carried out versus   SCE.

Notsu and Tatsuma ²⁰⁸ performed simultaneous determination of catechol and р-cresol by using tyrosinase electrode and a tyrosinase peroxidase enzyme electrode on the basis of differences of two phenolic compounds between the two enzyme electrodes. Stanca et al ²⁰⁹ , developed biosensor electrode for the determination of phenol and l- tyrosine.

III. A. 7. Chlorhexidine:

Chlorhexidine (Table I), is used either as the diacetate, digluconate or dihydrochloride salt.  It is most active in the pH range of 5-8.  It is generally stable in aqueous solutions but breaks down at elevated temperatures to produces р-chloroaniline especially in alkaline medium ²¹ . The reported methods of analysis of this compound can be classified as follows:

III. A. 7. a. High performance liquid chromatographic methods:

A RP - HPLC method for the determination of chlorhexidine, terbinafine HCl, and triamcinolone acetonide acetate in ointment was described by Ji et al ²¹⁰ .   Chlorhexidine in Kangtaizuo washing solution containing miconazole nitrate was determined utilizing RP-HPLC with UV detection ²¹¹ .

A stability-indicating HPLC assay for the determination of chlorhexidine in pharmaceutical products was developed by Ha and Cheung ²¹² . The method was also applied to study the hydrolytic pathway of the compound. Eleven impurities were isolated from chlorhexidine gluconate solution under various stress conditions and identified using chromatographic (HPLC and TLC) and spectrometric methods ²¹³ .

III. A. 7. b. Flow injection analysis:

A FI extraction–spectrophotometric method for micro determination of chlorhexidine in pharmaceutical products was devolped by Perez- Ruiz et al ²¹⁴ . The method is based on the formation of ion-pair between the compound and acid dyes such as bromophenol blue or bromothymol blue followed by measurement spectrophotometrically. A FIA procedure for the determination of chlorhexidine was described by Calatayud et al ²¹⁵ . The method is based on ion pair formation with thymol blue followed by turbidimetric detection.

III. A. 7. c. Spectrophotometric methods:

The content of chlorhexidine gluconate in several formulations was determined using UV spectroscopy ^216-218 , the use of colorimetric methods in the analysis of chlorhexidine gluconate in pharmaceutical preparations was demonstrated by ion-pair-formation with acidic dyes ^219,220 .

III. A. 7.  d. Electrochemical methods:

Polarography and differential pulse adsorptive stripping voltammetric methods were also reported for the determination of this compound ^221-223 .

III. B.  Analysis of Antioxidants preservatives:

Antioxidants are substances added to pharmaceutical products and food to inhibit their oxidation. They are commonly used in pharmaceutical products to protect the drug and /or excipients from autoxidative deterioration ^{1, 7, 224} . Although a large number of compounds have been shown to be useful as antioxidants, relatively few are used in pharmaceutical products. This is largely due to the unknown toxicity of many of these compounds and the great expenses required for the toxicological studies required approving the safety of the antioxidant for use. Antioxidants are classified into three groups; true oxidants, reducing agents and antioxidant synergists (Table II).

Oxidation reactions play an important role in the decomposition of pharmaceutical products and are considered to be a prime cause of product instability ²²⁵ . Oxidation is usually mediated through reaction with atmospheric oxygen under ambient conditions, a process commonly referred to as     autoxidation ^{226, 227} . Autoxidation is sometimes referred to as spontaneous process because it is often initiated by trace amount of impurities which may not be easily identifiable in the reaction mixture such as metal ions ²²⁸ [cupric, chromic, ferrous and ferric] or hydroperoxide. Also acids, bases, light, radiation, heat and some free radicals such as azo compounds may catalyze oxidation reactions. It is now well accepted that, most autoxidations are free-radical chain reactions. Free radicals are atoms or molecules that possess an unpaired valence electron ²²⁹ . Table (III) represents some functional groups subject to autoxidation.

Table (III): Some Functional Groups Subject to Autoxidation:

III. B. 1. Phenolic Antioxidants:

Phenolic antioxidants include, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tocopherols (Vitamin E), propylgallate (PG), 4-hydroxy-methyl-2,6-di-tert-butylphenol, tert-butyl-hydroquinone (TBHQ) and 2,4,5-trihydroxy-butyrophenone (THBP). Several assay methods have been reported for the analysis of phenolic antioxidants whether in pharmaceutical products. Some of these methods were discussed under antimicrobial preservatives. The reported methods of analysis of phenolic antioxidants can be classified as follows:

III. B. 1. a. Capillary electrophoresis methods:

Summanen et al ²³⁰ , have utilized CE for the resolution and determination of nine phenolic antioxidants; using uncoated fused silica capillary column, and an applied voltage of 22 kV. Delgado- Zamarreno et al ²³¹ , Saifar et al ²³² and Abidi and Rennick ²³³ applied the similar procedures for the analysis of phenolic antioxidants at different voltage. In all studies, the detection was carried out using UV detector.

Boyce and Spickett ²³⁴ reported a MECC method for the determination of phenolic antioxidants and other preservative (BHT and dodecyl gallate) in cosmetics.

III. B. 1. b. High performance liquid chromatographic methods:

Several experimental studies have been conducted to determine phenolic antioxidants in food and pharmaceutical products by chromatographic methods. As shown in the literature, there are several reviews for the determination of phenolic antioxidants. Robards and Dilli ²³⁵ reviewed the chromatographic and spectrophotometric methods for the determination of phenolic antioxidants. Another review authored by Abidi ²³⁶ focused on the various techniques for the isolation, purification, chromatographic separation and detection of tocopherols using normal and reversed phase HPLC was also reported. HPLC methods that have been used for the determination of phenolic antioxidants (PG, BHA, BHT, THBP, TBHQ and others) were reviewed by Karovicova and Simko ²³⁷ .

Yagoubi et al ²³⁸ , described a RP-HPLC procedure for the determination of phenolic antioxidants in pharmaceutical products. The Separation was carried out over Spherisorb column and the detection was carried out using UV-VIS spectrophotometer at 280 nm.

Direct injection of micro-emulsions oils in a micellar mobile phase was applied to HPLC with Spherisorb ODS-2 column, for the determination of synthetic antioxidants such as PG, dodecyl gallate, BHA, TBHQ, THBP and octylgallate. The detection was carried out using UV-VIS spectrophotometer at 284 nm ²³⁹ . Boussenadji et al ²⁴⁰ , described a microbore liquid chromatography with electrochemical detection for the determination of phenolic antioxidants in pharmaceutical products.

a-Tocopheryl acetate, PG., and butyl-4-methoxyphenol, either alone or combined with other antioxidants, in pharmaceutical products were determined by RP-HPLC. The detection was carried out by UV-VIS detector ^241-252 or fluorimetric detector ^{253, 254} . Also, a-tocopherol was determined by reversed phase-HPLC with coulometric detection ^255-257 . BHA, BHT and PG were determined by RP- HPLC with UV detection ²⁵⁸ or amperometric detection ²⁵⁹ Also, normal phase-HPLC with UV detection ²⁶⁰ and amperometric detection ²⁶¹ was reported.

a-Tocopherol, a-tocopheryl acetate and retinol were concentrated on C18, packed solid phase extraction cartridge then analyzed with HPLC using methanol/acetonitrile (3:7) as mobile phase and UV detection at 290nm ²⁶² .

Masuda et al ²⁶³ , applied supercritical-fluid chromatography to determine           a-tocopherol in ointment. Reversed phase-HPLC was used also for the determination of phenolic antioxidants in cosmetics and pharmaceutical products ^264-267 .

Other HPLC methods such as high performance capillary gel electrochromatography ²⁶⁸ , HPLC coupled with TLC ²⁶⁹ , micellar liquid chromatography ²⁷⁰ , capillary HPLC with amperometric detection ²⁷¹ and others ^{272, 273} were published for the determination of phenolic antioxidants.

III. B. 1. c. Gas Chromatographic methods:

BHA, BHT and TBHQ were also determined by gas chromatographic technique. Phenolic antioxidants were methylated by mixing with 10% methanolic tetramethyl ammonium hydroxide, followed by water and 0.2% tetra-decanol. The extract was concentrated and subjected to gas chromatography on a fused-Silica column, coated with CP-SIL8CB ²⁷⁴ .

Also BAH, BHT, t-butylhydroquinone and a-tocopherol were determined by gas chromatography on XAD-7 SPE column with nitrogen as a carrier gas and FID ²⁷⁵ .

a-Tocopherol, in pharmaceutical products of retinol, was concentrated with solid phase extraction on C18-SPE cartridge, followed by silylation and then determined by gas chromatography using FID ²⁷⁶ . 

The antioxidants; 4-alkyl-2,6-di-t-butylphenols [alkyl=methyl or butyl] were dervitized with pentafluorobenzoyl chloride in toluene in the presence of dry sodium hydroxide and benzyltriethyl ammonium chloride as a catalyst. The resulting solution was analyzed by gas chromatography on a glass column packed with 3% of SP-2100 with FID ²⁷⁷ .

III. B. 1. d. Thin layer chromatographic methods:

Thin layer chromatographic methods were reported for the identification and estimation of the activity of phenolic antioxidants in pharmaceutical products. Planar chromatography coupled with photodensitometric detection was described by          El-Mansouri et al , for identification of phenolic antioxidants in pharmaceutical products ²⁷⁸ . The products were dissolved in toluene, dried and extracted with methanol. The methanolic extract was applied to a RP-HPTLC, using acetonitrile-methanol-tetrahydrofuran mixture and detected photodensitometrically. Also              a preliminary study of the retention of some phenolic antioxidants on RP-TLC plates was described by Dimov ²⁷⁹ .

Both individual and total antioxidants (t-butyl-4-methoxyphenol, 2,6-di-t-butyl-p-cresol and santoquin) were determined by TLC and GC methods ²⁸⁰ . Samples were analyzed by TLC on Kieselgel-60 plates using n-heptane-ethyl octane as a mobile phase and detected spectrophotometrically.

a-Tocopherol in capsules was determined by combination of TLC and spectrophotometry ²⁸¹ . Other TLC methods for the analysis of this group of compounds were also reported ^{282, 283} .

III. B. 1. e. Flow injection analysis:

A flow–injection biamperometric method for the determination of gallic acid and tannic acid in pharmaceutical products was developed by Zhao et al ²⁸⁴ . The method is based on the electrocatalytic oxidation of pyrogallol compounds at one pretreated platinum electrode and the reduction of platinum oxide at the other pretreated electrode to form a biamperometric detection system with applied potential difference of 10 mV.

A flame ionization detector system was developed for the determination of BHA and lovastatin in tablets with UV and electrochemical detection ²⁸⁵ .

An amperometric flow-injection method for the determination of BHA, TBHQ and their mixture was described by Garcia and Ortiz ²⁸⁶ _. The authors utilized 4-hydroxybenzaldehyde/formaldehyde polymer modified electrode and applied it for the quantification of antioxidants in cosmetics.

Also, a flow-injection method with amperometric detector was described for the determination of the BHA and BHT ²⁸⁷ .

III. B. 1.f. Spectrophotometric methods:

Robards and Dilli ²³⁵ reviewed the spectrophotometric methods for the determinations of phenolic antioxidants up to 1987’s. a-Tocopherol was determined by formation of a ternary complex with copper (II) chloride and neocuproin in acetate buffer. The formed color was measured at 450 nm ²⁸⁸ . Pranjothy ²⁸⁹ described a direct UV-spectrophotometric method for determination of α-tocopherol in pharmaceutical products. He extracted α-tocopherol from the products into carbon tetrachloride and measured at 287 nm.  Also, Third and fourth dervative spectrophotometry was also applied by Ivanovic et al ²⁹⁰ , for the determination of certain phenolic antioxidants such as propyl gallate, t-butyl-4-methoxy-phenol and 2,6-di-t-butyl-p-cresol.

III. B. 1.g. Chemiluminesce methods:

Tocopherol was assayed by the quenching effect of the anthracene-sensitized electrochemi-luminescence ²⁹¹ . A chemiluminescene biosensing system for antioxidants such as propyl gallate was developed based on luminol and haematin co-immobilized on a cellulose membrane disc ²⁹² . BHA and PG were determined simultaneously by coupling stopped-flow mixing technique and diode-array detection ²⁹³ . The method is based on the reaction between antioxidants and 3-methylbenzothiazolin-2-one hydrazone in the presence of Ce (IV) and the reaction was monitored at 442 and 486 nm using a diode-array detector.

III. B. 1.h. Fluorimetric methods:

Lopez et al ²⁹⁴ , determined phenolic compounds as natural antioxidants by fluorescence method. They measured the antioxidant effect on the decline of            b-phycoerythrin fluorescence induced by a peroxyl radical generator, 2,2`-azobis     (2-amidino-propane) dihydrochloride with emission and excitation wavelengths of 575nm and 545nm respectively. The method was a modification of the method previously described by Cao and Prior ²⁹⁵ .

III. B. 1. i. Electrochemical methods :

Martin et al ²⁹⁶ , applied partial least squares regression to differential pulse voltammograms for the simultaneous determination of phenolic antioxidants (PG and 2-3-t-butyl-4-methoxy phenol. The voltammetric behavior of BHA, BHT, PG and TBHQ at a glassy carbon electrode in solution of 0.1 M perchloric acid containing 1% methanol had been investigated ²⁹⁷ . De la Fuente et al ²⁹⁸ , described the voltammetric determination of TBHQ and BHA at a polypyrrole electrode modified with nickel phthalocyanine complex as electron mediator.

Galeano-Diaz et al ²⁹⁹ , and Agui et al ³⁰⁰ , studied the voltammetric behavior phenolic antioxidants (BHA, BHT and PG), and their simultaneous determination in pharmaceutical products. Asuncion-Ruiz et al ³⁰¹ , utilized carbon-paste electrode modified with nickel phthalocyanine for the voltammetric determination of the antioxidant BHT.

III. B. 2. Analysis of reducing agents:

III. B. 2. a.  Ascorbic acid (ASA) and its salts:

As shown in the literature, there are several reviews for the determination of ascorbic acid. Al-Meshal and Hassan ³⁰² in 1982 reviewed the different methods of analysis of ascorbic acid. Another review by Pachla et al ³⁰³ , in 1985, focused on the various techniques for determining ASA in pharmaceuticals, biological samples and food products. Ascorbic acid is an important vitamin having a chemical structure that justifies its classification as a carbohydrate (Table II). It is rapidly oxidized to dehydroascorbic acid by oxygen and metal ions, and then the dehydroascorbic acid can be further oxidized to diketogu lconic acid as follows:

Scheme 1: Oxidation Mechanism of Ascorbic acid.

It is difficult to discriminate between the methods of analysis of ascorbic acid as active ingredient or as antioxidant preservatives these methods can be classified as follows:-

III. B. 2. a. i. Capillary electrophoresis methods:

Utilization of CE for the determination of ASA and biotin in pharmaceutical products was investigated by Schiewe et al using diode array detector ³⁰⁴ . Also, ASA and other vitamins were analyzed by CE using fused-silica column. The detection was carried out using amperometric detector ³⁰⁵ . Yin and Wu ³⁰⁶ determined the active constituent in vitamin C tablets by MECC, using fused-silica capillary column, sodium borate and sodium deoxycholate at pH range 9-11 as running buffer. The detection was carried out using UV-VIS spectrophotometer at 254 nm. Hu et al ³⁰⁷ , used MEKC with amperometric detection for the determination of nicotinamide, pyridoxine and ascorbic acid in pharmaceutical products and foods.

III. B. 2. a. ii. High performance liquid chromatographic methods :

The degradation of ASA stored in parenteral nutrition and pharmaceutical products occurs initially by oxidation with oxygen and is catalyzed by trace elements, in particular copper ions (Scheme 1). For this reason, Mueller ³⁰⁸ described a rapid sample extraction procedure for the determination of ascorbic acid by RP-HPLC in multivitamin mineral tablet containing interfering copper.

Also, stability-indicating reversed-phase HPLC method was used with UV detection at 278 nm by Gibbons et al ³⁰⁹ . They studied the effect of temperature and trace elements on the anaerobic degradation of dehydroascorbic acid in parenteral nutrition mixtures. Irache et al ³¹⁰ , described a RP- HPLC method for the determination of ASA and the antioxidant synergists such as citric acid, tartaric acid, lactic acid and EDTA, in some pharmaceutical products and cosmetics. They utilized Spherisorb-ODS column. The detection was carried out at 210 nm.

Comparative HPLC and voltammetric studies were described by Esteve et al , for the determination of ASA in infant products ³¹¹ . The results concluded that the two methods could be used for the routine analysis of ASA.

Mori et al ³¹² , described HPLC with fluorimetric detection using 4,5-dimethyl-o-phenylenediamine as reagent for the analysis of total ASA and dehydro-ASA .The method is based on the oxidation of all ASA to dehydro-ASA prior to the analysis. Yang ³¹³ utilized HPLC for the determination of ASA in tablets using amino column, buffered aqueous acetonitrile as mobile phase and UV detection. Austria et al ³¹⁴ , investigated the stability of the antioxidants (ASA, ascorbyl palmitate and magnesium ascorbyl phosphate) in standard solutions and topical formulations by RP- HPLC.

Kinetic HPLC studies for the analysis of ASA based on the quenching of the chemiluminescence intensity of the reaction between luminol and superoxide anion radical was presented by Toyo'oka et al ³¹⁵ .

Ascorbyl palmitate and other phenolic antioxidants were determined by HPLC with UV detection at 255 nm for ascorbyl palmitate and at 280 nm for other phenolic antioxidants ³¹⁶ .

III. B. 2. a. iii. Flow Injection analysis:

The high flexibility of FI has fostered the development of a wide variety of multi-determinations based on a number of chemical systems. However, the assemblies involved are occasionally high complex and use several detectors. So, many FI methods have been reported for the determination of ASA in pharmaceutical preparations, food products and biological samples using different detection systems ³¹⁷ .  Most of the published methods are based on oxidation of ASA to DHAA followed by UV measurements ^318-320 or by coupling with certain reagents ^321-325 and resoluting over different columns.

Recently; Legnoerova et al ³²⁶ , utilized a sequential injection technique coupled with a solid phase extraction for the simultaneous determination of ASA and rutin tri-hydrate in pharmaceutical products.

III. B. 2. a. iv. Spectrophotometric methods:

The spectrophotometeric methods for determination of ASA in pharmaceutical products, food, and biological samples up to 1998 were reviewed by Arya et al ³²⁷ .      

A simultaneous spectrophotometric method for the determination of ascorbic acid, quercetin and rutin in pharmaceutical products (tablets and capsules) was reported by Hassan et al ³²⁸ .  The authors utilized a computer program based on kalman filter multivariate calibration.

Solid phase extractions coupled with UV spectrophotometer were applied for determination of ASA in pharmaceutical products ³²⁹ . The measurements carried out at 267 nm.        

First derivative spectroscopy was utilized by Dogan and Duran ³³⁰ or the simultaneous spectrophotometric determination of ASA and aspirin. Surmeian ³³¹ applied the same procedure for the determination of ASA and pyridoxine hydrochloride in dosage forms ³³² . ASA and Salicylic acid in pharmaceutical products were also determined by first derivative spectroscopy, using the zero-crossing method at 256 nm for ASA ³³³ . Colorimetric methods were also reported for the determination of ASA in pharmaceutical products by Arya et al ^{334, 335} .

A kinetic spectrophotmetric method based on measurement of the decrease in absorbance of the developed color between Fe (III) and o-phenanthroline monohydrate was described by Issopoulos and Salta ³³⁶ .

III. B. 1. a. v.  Chemiluminescence methods:

Chemiluminescence methods were also reported for the determination of the antioxidant activity of ASA in pharmaceutical products.  Papadaopoulos et al ³³⁷ described a sensitive and simple method for measuring the antioxidant activity which based on antioxidant-dependent quenching of the chemiluminescence generated from photolysed acridine and methanolic potassium hydroxide. This method was used to evaluate the antioxidant activity of additives as ASA, citric acid and tartaric acid by measuring the inhibition of chemiluminescence at a gradient of antioxidant concentrations.

Stadnichuk et al ³³⁸ described a chemiluminescence method for determination of ascorbic acid in pharmaceuticals using an aqueous solution of the drug containing borate buffer of pH 10.2, luminol, Mn(II) triethanolamine complex and peroxymonosulfuric acid. Zhu et al ³³⁹ determined ASA in tablets and injections preparations by chemiluminescence spectroscopy after treatment with potassium permanganate in sulfuric acid medium and the chemiluminescence intensity was measured. A spectrophotometric method for determination of ASA depending on the reaction with 2-cyanoacetamide at pH 3 was developed by Yang   et al ³⁴⁰

III. B. 2. a. vi. Electrochemical methods:

The determination of ASA has been a major target of electroanalytical research. It is almost too difficult to determine this compound electrochemically by direct oxidation on a convenient electrode because of its large over potential and fouling by the oxidation products ³⁴¹ . Therefore, some chemically modified electrodes (CME) with active mediators immobilized at the electrode surface have been used for the catalysis of electrooxidation ³⁴² .

CME of polypyrool / ferrocynide films modified carbon paste electrode has been used for the determination of ASA ³⁴³ . Also chloine and acetylcholine modified glassy carbon electrode was applied for the simultaneous determination of dopamine, serotonin and ASA in pharmaceutical products ³⁴⁴ .

Differential pulse voltammetery was applied for the determination of ASA with CME (ferrocene-L-cysteine film coated electrode) ³⁴⁵ . Cyclic and differential pulse voltammetric methods for the determination of ASA, BHA, BHT and propyl gallate were recorded with carbon-paste electrode or graphite PTFE disk composite electrode as the working electrode and Ag/AgCl as reference electrode and a gold counter electrode ³⁴⁶ .

Electrochemical behavior of ASA at a poly (glutamic-acid)-coated glassy-carbon electrode was studied by cyclic voltammetry ³⁴⁷ Other methods include the use of amperometric determination of ASA on screen-printing ruthenium dioxide electrode ³⁴⁸ or on ruthenium (III) diphenyldithiocarbamate modified carbon paste electrode ³⁴⁹ or using polarographic method ³⁵⁰ . Ijeri et al ³⁵¹ studied the voltammetric behavior of ascorbic acid on a chemically modified electrodes based on use of macrocyclic compounds by electrokinetic oxidation. This method was used for determination of ASA in multivitamin-multimineral pharmaceutical preparations.

Blazheev'skii et al ^352, described the amperometric determination of ascorbic acid in pharmaceuticals together with acetylcysteine. An aqueous solution of powdered preparation in sulphuric acid was titrated against perbenzoic acid with amperometric detection.

III. B. 2. a. vii. Titrimetric methods:

ASA and cysteine in eye drops products were determined by potentiometric titration. The aqueous sample solution in potassium dihydrogen phosphate was titrated potentiometrically against diperoxyadipic acid ³⁵³ .

Riyazuddin and Nazer ³⁵⁴ described a potentiometric method for determination of ASA in pharmaceutical products, using copper based mercury film electrode. 

III. B. 2. b.  Sulfites:

This group of chemicals includes: sulfite, bisulfite and metabisulfite (Table II). They are strong reducing agents and are widely used as antioxidants in solutions especially those containing drugs which are readily oxidized to form highly coloured decomposition products. They are also used as antimicrobial preservatives in acidic solutions and syrups ²¹ . Several methods have been reported for the determination of sulfites in pharmaceutical products; these methods can be classified as follows:

III. B. 2. b. i.  Capillary Electrophoresis method:

In recent years, CZE with indirect UV detection (negative peaks were recorded) have been successively applied for the analysis of inorganic anions. Geiser et al ³⁵⁵ , described simultaneous CE with indirect UV method for the determination of metabisulfite and sulphite in pharmaceutical products. Pyromellitate was used as chromophoric agent with indirect measurement at 255 nm.

III.B. 2. b. ii. High performance liquid chromatographic methods :

Parkin ³⁵⁶ described stability-indicating HPLC procedures for the determination of the degradation products of PMN and sodium metabisulfite in eye drop formulations. Herbranson et al ³⁵⁷ , developed a high performance ion chromatographic (HPIC) method for the determination of sodium metabisulfite in parentral products. HPLC was also applied for the determination of metabisulphite in antrheumatic injections ³⁵⁸ and in penicillins ³⁵⁹ with or without derivatization.

III. B. 2. b. iii.  Flow injection analysis :

A FI-coulometric flame ionization analysis of metabisulfite with iodine in the presence of starch was developed by Taylor et al ³⁶⁰ .       

Masson and Townsend ³⁶¹ described FI method for the determination of sulfite and sulfite oxidase. Formaldehyde was used to stabilize the solution which was determined by injection into a flowing stream of pH 8.5 (phosphate buffer) using a mini-column with amperometric detection. Lin and Hobo ³⁶² described a FI with chemiluminescent detection for selective determination of sulfite.

Sulfite in capsules containing vitamin K was determined by chemiluminescence in flow system ³⁶³ . The method is based on the fact that a weak chemiluminescence is produced by autoxidation of sulfite in the presence of rhodamin 6G, immobilized on a cation exchange column. Tween 80 micelles showed a strong enhancement on this weak chemiluminescence. Also, Huang et al , described the same procedure for determination of two sulfite containing drugs (menadione sodium bisulfite and analgin) in pharmaceutical products ³⁶⁴ .

III. B. 2. b. iv.  Spectrophotometric methods:

Colorimetric methods were reported for the determination of sodium bisulfite in parenteral mixture or in inhalation solutions ^365,366 . Hussain et al ³⁶⁷ , described the reaction of cis-platinum (antitumor agent) with pharmaceutical additives as sodium bisulfite (antioxidant) in acetate buffer with absorbance at 280 nm. An automated method for the determination of sulfite and sulfur dioxide by cool flame molecular emission spectrometry was described by Arowolo and Cressor ³⁶⁸ . The method is based on the reduction of both compounds to hydrogen sulfide with sodium tetrahydroborate (III) and the intensity of the blue diatomic sulfur emission generated is measured at 384 nm.

III. B. 2. b. v.  Titrimetric methods:

Direct ^369-370 and indirect titrimetric methods ³⁷¹ with potentiometric end point detections were described for the determination of metabisuphit using different oxidizing agents.

III. 2. 3. Analysis of synergists antioxidants (EDTA and its salts):

Ethylendiaminetetraacetic acid (EDTA), is an organic complexant and its chemical structure is shown in table II.  It is usually symbolized by H ₄ Y, because of its acidic functions. The values of the four corresponding pK _a are 2, 2.8, 6.2 and 10.3.

EDTA and its salts have been used in a wide range of industrial, pharmaceutical and agriculture applications. It is the most effective chelator for use in pharmaceutical products.  It is not only used as a preservative but also as antioxidant synergists and it is added to compounds which usually have little antioxidant effect themselves but probably enhance the action of other antioxidants by reacting with heavy metal ions which catalyze oxidation. In the literature, several publications deal with the measurement of EDTA and its related compounds, in most of these studies, metal cations are added to the sample prior to the determination. Sillanpaa and Sihvonen ³⁷² reviewed the different methods for the determination of EDTA and related compounds.  Another review was published by Belal and Al-Badr ³⁷³ in 2002, focused on the various techniques for the determination of EDTA in pharmaceutical products.

III. B. 3. a. Capillary electrophoresis methods:

CE was utilized for the determination of Fe(III)-EDTA chelate and EDTA itself in solutions. EDTA was complexed with Ni (II) salt prior to analysis, separated in a fused-silica capillary filled with a borate buffer of pH 8.5 and detected at 214nm using UV detector ³⁷⁴ .

Owens et al ³⁷⁵ , studied the analysis of nitrilotriacetic acid and EDTA mixtures by CE. Various metal ions [Ca(II), Co(II), Ni(II), Cu(II), Zn(II), Cd(II), Pb(II) and Fe(III)] were added to the samples and an electrolyte composed of Na ₂ HPO ₄ was used. The authors reached a detection limit (UV detection at 185 nm) of 2 and 5 um. The method was applied to demonstrate speciation in complex nutrient media.

Padarauskas and Schwedt ³⁷⁶ separated the complexes formed between EDTA and ferric with electrolyte composed of acetate buffer at pH 4, with UV detection at 254 nm.

III. B. 3. b. High performance liquid chromatographic methods:

HPLC coupled with mass spectrometry (MS) via electrospry interference has exhibited a powerful capacity in the measurement of EDTA, in the form of its iron complex. The complex was eluted through a stationary phase under slightly acidic conditions and detection was performed by mass spectrometry in the single ion monitoring mode ³⁷⁷ .

EDTA in injection forms was determined by RP-HPLC. The method is based on the formation of ion pair complex with Cu (II) ions. The chromatographic separation of the complex was performed on C8 column. The mobile phase consisted of acetonitrile- tetrabutylammonium hydroxide of pH 7. The detector was operated at 300 nm ³⁷⁸ . Similar procedures were described for the determination of EDTA in ophthalmic products, the complex was resolved using tetrabutyl ammonium hydroxide-phosphoric acid-methanol at the same pH as mobile phase and detection was carried out at 254nm ^379-380 or 250 nm ³⁸¹ . Shi and Chen ³⁸² applied the same principle for the determination of this compound in canned food products.

Tran et al ³⁸³ , described the utilization of Fe(III)  for the complex formation and the determination of EDTA in ophthalmic cleansers. The analysis was performed using RP-HPLC with C18 column, tetrabutylammonium hydrogen sulfate in water as a mobile phase and detection was at 254 nm. EDTA in vancomycin products (containing vancosin hydrochloride) was determined by HPLC, through formation of complex with  Fe(III) which was separated on a column of ultrasphere ODS, with linear gradient elution and detection at 260 nm ³⁸⁴ .

Stalberg and Arvidsson ³⁸⁵ described HPLC method for the determination of EDTA as its metal complex on a porous graphitic carbon column with aqueous mobile phase containing ethylene glycol and ferric sulfate, and the complex was measured at 270 nm.

III. B. 3. c. Spectrophotometric methods:

Several spectrophotometric methods have been reported for the determination of EDTA. Most of these methods are based on the formation of ternary complex between EDTA and metal cations followed by coupling with another chelating agent.  For example, EDTA in ophthalmic formulations, was determined using magnesium sulfate and arsenazo-I indicator ³⁸⁶ . Cu(II)-EDTA sequestrate was treated with 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline in chloroform. The organic layer was separated and the absorbance was measured at 477 nm. Also, Fe(III)-EDTA sequestrate which would form a chromophore with 4,7-diphenyl-1,10-phenanthroline disulphonic acid was used and the absorbance  was measured at 535 nm ³⁷⁸ .  Cu(I) ternary complex was  produced by the reaction of  Cu(I) , EDTA and pyridine-2-carboxaldehyde at pH 8.5 and the absorbance was measured at 475 nm ³⁸⁸ . Another colored ternary complex method was developed by Paris et al ³⁸⁹ , where EDTA was mixed with copper sulfate and 1-(2-pyridylazo)-2-naphthol and the color produced was measured at 551 nm.

Kinetic spectrophotometric technique was also applied for the determination of micro amounts of EDTA in rinsing solution for contact lenses ³⁹⁰ . The method is based on catalytic acceleration of the reaction between butylrhodamine and potassium dichromate at pH 12.5. The decrease in absorbance was measured at 560nm.

III. B. 3. d. Atomic Absorption Spectrophotometry:

EDTA in streptomycin products was determined by an atomic absorption spectroscopic (AAS) method based on its complexation with nickel ions ³⁹¹ . The          Ni-EDTA sequestrate was directly measured by atomic absorption spectroscopy.

EDTA was determined by flame atomic spectroscopy using an ion-exchange resin adopting on-line technique through its reaction with copper (II) ^392. The eluted copper (II) was determined by flame atomic spectroscopy.

Belal et a ³⁹³ , described AAS method for the determination of EDTA in some eye drop products. This method is based on the reaction of EDTA with calcium (II) or magnesium (II) ions. The formed complexes were separated using cation exchange resin and determined by flame atomic absorption spectroscopy. Guclu et al ³⁹⁴ , developed a combined visible spectrophotometric and atomic absorption spectroscopic methods for the analysis of trace metal, EDTA and EDTA-metal mixture in solutions.

III. B. 3. e. Fluorimetric methods :

The ternary complex formation was also utilized for the fluorimetric determination of EDTA. For example the reaction of lanthanide ions [La(III) and Y(III)] with 7-(1-naphthylamineazo)-8-hydroxyquinoline-5-sulfonic acid has been exploited for the fluorimetric determination of EDTA. The fluorescence was measured at 525/350 nm ³⁹⁵ . A similar procedure utilizing terbium–salicylate complex was applied for the determination of EDTA in cell culture medium ³⁹⁶ .

The reaction of Ca(II) with EDTA in the presence of N-cyclohexyl-3-aminopropane sulfonic acid (pH 10) was described for the fluorimetric determination of EDTA at 485/535 nm ³⁹⁷ .

III. B. 3. f. Electrochemical methods:

The polarographic technique has been applied for the determination of EDTA in pharmaceutical products. Belal et al ³⁹⁸ , developed a polarographic method for the determination of EDTA in certain pharmaceutical products, as eye drop preparations and cevarol ampoules, after complex formation with europium (III) chloride. Copper (II) complex with EDTA was also used for polarographic determination of EDTA in solutions ³⁹⁹ .

Cadmium (II) or zinc (II) complexes in an ammonia buffer of pH 9.5, were used for the determination of EDTA in ophthalmic products. The decrease in the peak height of the metal ion using differential pulse polarographic mode was equivalent to the amount of EDTA ⁴⁰⁰ . A potentiometric method for the determination of EDTA involved the use of a urease-based inhibition biosensor was also reported ⁴⁰¹ .

III. B. 3. g. Titrimetric methods:

Danel'yants et al ⁴⁰² , determined EDTA in injections (containing morphine, codiene, narcotine, papaverine and thebaine) by titration with ferrous ammonium sulfate in the presence of ammonium persulfate using sulphonsalicylic acid solution as indicator.

 

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{mospagebreak title=About Authors }

About Authors:

Khairi M.S. Fahelelbom ^a , Yasser El-Shabrawy ^b

^a Faculty of Pharmacy and Health Science, Ajman University of Science and Technology, Fujairah Campus 2202, United Arab Emirates.

* Corresponding author

E-Mail: Khairi_f@hotmail..com

Fax: +971-9-2227644, Phone: +971-50-4492416

^b Faculty of Pharmacy and Health Science, Ajman University of Science and Technology, Ajman 346, United Arab Emirates