Rp-HPLC Ion Pair Method Development, Validation And Stability Indicating Assay For Famciclovir
By - 04/29/2007
(1 vote)
K.V.Subrahmanyam
Drug Profile :
The Famciclovir 1-4 a synthetic 6-deoxy, diacelyl aster of Penciclovir, is an antiviral prodrug that is active against herpes simplex types 1 (HSV-1) and 2 (HSV-2) and varicella zoster virus (VZV). Famciclovir has no antiviral activity but is metabolized to Penciclovir, a potent and selective antiviral agent.
The limited oral absorption of the acyclic guanine derivative, Penciclovir (9-(4hydroxy-3-hydroxymethylbut-1-yl) guanine; prompted a search for orally bioavialable prodrugs. Famciclovir (9-(4-acetoxy-3 acetoxymethyl but -1-yl)-2 -aminopurine, it is a well-absorbed oral prodrug of Penciclovir. It is the diacetate ester of 6-deoxyPenciclovir. It is the 6-deoxy precursor of Penciclovir.
Molecular Weight: 321.33
Molecular Formula : C 14 H 19 N 5 O 4
C – 52.33%
H – 5.96%
N – 21.79%
O – 19.92%
Chemical Structure :
Antiviral Activity :
Famciclovir 1 has activity against herpes simplex types 1 and 2, varicella-zoster virus and Epstein-Barr virus in vitro. Famciclovir is also active in vitro against other herpesviruses, e.g. cytomegalovirus (CMV).
Mode of Administration and Osage:
Availability :
Famciclovir is available for oral administration in 125, 250 mg and 500 mg tablets.
Herpes Zoster
Three 250mg tablets once daily for seven days or altematively one 250 mg tablet three times daily for seven days. If the tablets are taken once a day they should be taken at approximately the same time each day. In immunocompromised patients, one 500mg tablet three times daily for 10 days. Treatment should be initiated as early as possible in the course of the disease, promptly after diagnosis. In the U.S. the recommended dosage is 500mg every 8 hours for 7 days. Therapy should be initiated promptly as soon as herpes zoster is diagnosed. No data are available on efficacy of treatment started greater than 72 hours after rash onset.
Herpes Simplex Intections
Recurrent gential herpes: The recommended dosage is 125 mg twice daily for 5 days. Initiate therapy at the first sign or symptom if medical management of a genital herpes recurrence is indicated. The efficacy of Famciclovir has not been established when treatment is initiated more than 6 hours after onset of symptoms or lesions.
Suppression of recurrent genital herpes : The recommended dosage is 250 mg twice daily for up to 1 year. The safety and efficacy of Famciclovir therapy beyond 1 year of treatment have not been established.
HIV-Infected Patients
For recurrent genital herpes simplex infection, the recommended dosage is 500 mg twice daily for 7 days in patients with reduced renal function, dosage reduction is recommended.
Summary of clinically relevant pharmacokinetics of Famciclovir
Good bioavailability of Penciclovir after oral administration of Famciclovir (41-75%). May be taken with food.
Plasma levels increase in patients with renal dysfunction, requiring dose adjustment. Famciclovir is metabolized in the liver and excreted predominantly in the urine.
Excretion And Distribution Of The Drug In Body :
Famciclovir undergoes extensive first-pass metabolism. The metabolic conversion of Famciclovir to Penciclovir is primarily through deacetylation within the intestinal wall producing the 6-deoxy derivative of Penciclovir followed by oxidation at the 6-position in the purine base by aldehydes oxidase within the liver to produce Penciclovir.
Penciclovir is excreted primarily by the renal route, by glomerular filtration as well as tubular secretion, with little or none of the parent compound Famciclovir being measured in the urine or blood. The terminal phase elimination half-life of Penciclovir is approximately 2h.
Mode Of Action :
Famciclovir is a prodrug of Penciclovir, the latter being the active antiviral compound upon its triphosphorylation within the infected cell. Famciclovir is the diacetyl 6-deoxy analog of Penciclovir, Penciclovir is phosphorylated by virus-specified TK to the monophosphate derivative, cellular kinases then convert Penciclovir monophosphate to the di-and triphosphate forms. Phosphorylation of Penciclovir occurs more rapidly in infected cells than acyclovir.
Biotransformation of Famciclovir in man (Reproduced from Vere Hodge et al., 1989)
Fig 3 Biotransformation of Famciclovir
Schematic representation of inhibition of the viral DNA synthetic pathway by Penciclovir in a herpesvirus-infected cell. (Reproduced from Perry and Wagstaff, 1995)
Chart I
Drug Interactions :
Concurrent use with Probencecid or other drugs significantly eliminated by active renal tubular secretion may result in increase plasma concentrations of Peniciclovir. The conversion of 6-deoxy Penciclovir to Penciclovir is catalyzed by aldehydes oxidase. Interations with other drugs metabolized by this enzyme could potentially occur.
Adverse Reactions :
The main side effects reported, were head ache, nausea, diarrhoea, fatigue, dizziness, abdominal pain, and dyspepsia. The incidence of adverse effects is similar in immunocompromised patients.
Introduction
Reversed-Phase Chromatography :
Two very advantageous characteristics of HPLC, its discriminating power and its ability to operate at room temperature or at low elevated temperature, would not contribute to the degradation of the analyte. It is further assumed that the API is of low molecular weight (<1000 Daltons ), organic in nature (versus inorganic), and not a biopharmaceutical. These restrictions apply to a large percentage of the pharmaceutical. These restrictions apply to a large percentage of the pharmaceuticals and enable them to be readily separated using reversed-phase HPLC, and sometimes with the aid of an ion-suppression agent, in roughly 85%of the applications.
Role Of The Column:
The HPLC column is the heart of the method, critical in performing the separation. The column must posses the selectively, efficiency, and reproducibility to provide a good separation. All of these characteristic are dependent on the column manufacture’s production of good quality columns and packing materials. Properties of the silica (backbone) such as metal content and silanol activity produced in the manufacturing and bonding process determine the properties of the finished bonded phase. A good silica and bonding process will provide the reproducible and symmetrical peaks necessary for accurate quantitation.
Commonly used reversed phases are C18 (octadecylsilane, USP L1), C8 (octylsilane, USP L7), phenyl (USP L11), and cyano (USP L18) (24). They are chemically different bonded phases and demonstrate significant changes in selectivity using the same mobile phase.
Selection Of The Chromatographic Method
Chart II
Role Of Flow Rate :
Flow rate, more for isocratic than gradient separation, can sometimes be useful and readily utilized to increase the resolution, although its effect is very modest. The slower flow rate will also decrease the column back pressure. The disadvantage is that when flow rate is decreased, to increase the resolution slightly, there is a corresponding increase in the run time.
Role Of Temperature :
While temperature is a variable that can affect selectivity, a, its effect is relatively small. Also, the k ' generally decreases with an increase in temperature for neutral compounds but less dramatically for partially ionized analytes. Snyder et al. (29) reported that an increase of 1 o C will decrease the k ' by 1 to 2%, and both ionic and neutral samples.
Role Of Ph :
pH is another factor in the resolution equation that will affect the selectivity of the separation. In reversed-phase HPLC, sample retention increase when the analyte is more hydrophobic. Thus when an acid (HA) or base (B) is ionized (Converted from the unionized free acid or base) it becomes more hydrophilic (less hydrophobic, more soluble in the aqueous phase) and less interactive with the column’s binding sites.
HA « H + +A -
B + H + « BH +
Hydrophobic Hydrophilic
(more retained on column) (less retained on column)
As a result, the ionized analyte is less retained on the column, so that the k ' is reduced, sometimes dramatically. When the pH =pK a for the analyte, it is half ionized, i.e., the concentrations of the ionized and unionized species are equal. As mostly all of the pH-caused changes in retention occur within ±1.5 pH units of the pK a value , to ensure practically 100% unionization for retention purposes. The pH range most often encountered in reversed-phase HPLC is 1-8, normally considered as low pH(i.e.,1-4) and intermediate pH(i.e.,4-8). Generally, at lwo pH peak tailing is minimized and method ruggedness is maximized. On the other hand, operating in the intermediate range offers an advantage in increased analyte retention and selectivity.
Role Of The Ion – Pair Reagent :
Initially, when deciding whether to select reversed-phase HPLC or reversed-phase HPLC with ion-pairing, a good rule of thumb is to consider the nature of the analyte of interest. If the sample is neutral, begin with reversed-phase; and if the sample is ionic, use ion-pairing. Thus reversed-phase HPLC and reversed-phase HPLC with ion-pairing are similar except that the latter contains an ion-pair reagent in the mobile phase to improve the selectively of ionic samples. The use of an ion-pair reagent is suggested only when separation is not adequate with reversed-phase HPLC. This is because using an ion-pair reagent introduces additional experimental parameters that need to be controlled, such as what ion-pair reagent o use and its concentration. Because of this added variable, reversed-phase HPLC should be utilized on any ionic analyte first before trying ion-pair reversed –phase HPLC. The solubility of the ion-pair reagent may also be affected depending on the organic solvent used in the mobile phase.
Table I
Listing of types of Ion-pair reagent and conditions of Use
|
Ion-pair reagent type and example |
Used for compound class |
pH of mobile phase |
Concentration of ion-pair reagent |
|
Alkyl sulfonates (sulfonic acid alkyl salts) |
Cationic samples (protonated bases) |
3.5 |
0.005M |
|
Octane sulfonate |
Basic compounds |
2.5 |
0.002M |
Method Validation :
General chapter <1225> states, “Validation is the process of providing documented evidence that the method does what it is intended to do.” In other words, the process of method validation ensures that the proposed analytical methodology is accurate, specific, reproducible, and rugged for its intended use.
Validation Parameters :
Prior to conducting validation studies it is imperative to decide which parameters are required to be studied. These parameters are termed “analytical performance characteristics” or sometimes “analytical figures of merit.”
Table II
Comparison of Analytical Parameters Required for Assay validation
|
FDA reviewer Guidance |
USP General Chapter <1225> |
ICH Q2A Guidelines |
|
Accuracy |
Accuracy |
Accuracy |
|
Precision Repeatability Injection Analysis |
Precision No |
Precision No |
|
Intermediate precision |
No |
Intermediate precision |
|
Reproducibility |
No |
No |
|
Specificity /selectivity |
Specificity |
Specificity |
|
Detection limit |
Detection limit |
Detection limit |
|
Quantitation limit |
Quantitation limit |
Quantitation limit |
|
Linearity |
Linearity |
Linearity |
|
Range |
Range |
Range |
|
No |
Ruggedness |
No |
|
Robustness |
Robustness |
Robustness |
|
System suitability sample solution stability |
System suitability |
System suitability |
Plan Of Work
- Gather/generate background information – obtain physico-chemical properties.
- Determine if special handling/treatment of sample is needed
- From physicochemical properties select detector l max.
- Select LC mode and perform initial runs
- Guesstimate separation parameters/isocratic or gradient mode
- Perform forced degradation experiments to challenge method
- Optimize separation conditions; peak R s , equation parameters
- Summarize methodology finalize documentation
- Validate method/ Transfer to control Laboratory.
Development Of An Assay For Famciclovir In Pharmaceutical Preparation By Hplc Ion Pair
Introduction :
Famciclovir 2 - [2 - (2 – amino – 9H – purin – 9 – y1) ethyl] trimethylene diacetate) is a modified nucleoside which shows strong activity against viruses of the Herpes group 9 . It derives from guanine, which is a constituent of purine. Recently, several methods have been reported for the separation of the nucleoside including High Performance Liquid Chromatography (HPLC) by using ion exchange packed chromatography column materials 10 . However, these methods are generally involved and give poor chromatographic peak shapes.
The problems of sensitivity are compounded by the possibility of the nitrogen-containing basic drugs and quaternary ammonium compounds causing problems in RP-HPLC, where peak tailing and extended or even total, retention is often observed. The peak tailing effects are most probably due to the presence of residual (non-reacted) silanol groups on the silica surface, the drug being present in the ionized form.
In order to improve the chromatographic properties of basic solution, a number of alternative methods have been suggested. Among these methods ion pair chromatography is widely used, especially in the bio-analytical area where there is likely to be a range of polarities present in the biological matrix of the drug and its metabolites. Although it is possible to increase the retention simply by decreasing the content of organic solvent in the eluent, the effect on the presence of non-ionic compounds, then an increase in retention can be controlled selectively by the addition of an anionic counter ion 9-20 .
In this work a reversed phase ion pair HPLC method was developed to separate Famciclovir from its major degradation product.
Experimental
The chromatographic system consisted of Shimadzu LC 20A pump (Touzart, Matignon, France) connected to a Rheoyden 7520 syringe loading sample injector valve fitted with a 20 mL sample loop, a variable wavelength UV detector (UV SPD 20 A) and a Shimadzu chart recorder. The column was a 250 x 4.6 mm, packed with Gemini ODS C18, 5 mm particle size (Phenomenox). Famciclovir was obtained from Cipla Limited, Daman . Famciclovir (VIROVIR) tablets were obtained from FDA.
Optimization Of Mobile Phase : Optimization of mobile phase was required to achieve resolution between Famciclovir an its degradation products while maintaining good peak symmetry and reasonable analysis time 21-23 . This was accomplished by investigating the effect of “counter ion type” and concentration, percentage organic modifier, mobile phase pH on capacity factor ( k ’).
Hplc Method Development Procedure For Famciclovir:
Detection Of Wave Lenth:
In setting up the conditions for development of the assay method, the choice of a detection wavelength was based on the scanned absorption spectrum for Famciclovir. The spectrum was scanned over the range of 200-400 nm and was obtained by measuring the absorption of 10 mg/mL solution of Famciclovir in mobile phase, prepared from a stock solution. The spectrum was obtained by using a 1cm silica cell and the reference cell contained mobile phase; as a result a wavelength of 314 nm was chosen.
Effect Of Organic Modifier On The Of Famciclovir Capcity Factor On C 18 Column:
In optimizing the separation of Famciclovir from its degradation products. In this first instance the effect of the organic modifier on the retention of was Famciclovir was examined on C 18 stationary phase (Fig. VI). This shows the dependence of the capacity factor (k’) of Famciclovir on the volume percentage of actonitrile (ACN), therefore the ODS stationary phase was chosen for this study and 5% ACN was sufficient to give reasonable retention time.
Graph : I
Effect Ph Of The Mobile Phase On The Capcity Factor And Peak Shape Of Famciclovir:
Following this, the effect of the pH of the mobile phase on the retention and peak shape for Famciclovir was examined (Fig. VII), where the influence of the pH of the mobile phase on k ’ is illustrated. It is clear from this figure that the capacity factor was found to increase on decreasing the pH values for ACN/H 2 O system. It was also observed that the pH for the optimum conditions is related to the pKa of famciclovir. At pH < pKa, the Famciclovir will be available in its maximum cationic form (HACN + ) which may interact with the counter ion form. At pH> pKa. The Famciclovir will be available in neutral form (HCN). The major limitation is the workable pH range and life time of the column. According to the results and the improvement in peak shape, and retention time, pH 2.5 was chosen for ACN/H 2 O- system prior to use.
Graph : II
Effect Of Concentration Of Counter – Ion On The Capcity Factor:
The effect of alkyl type and concentration of the counter-ion on the capacity factor was studied and the results are given in Fig.VIII. As can be seen, the k ’ was found to increase with increase in alkyl type and concentration of the counter-ion. Therefore C 8 H 17 SO 4 Na at 0.002 M was chosen prior to use. The combination of all these parameters is demonstrated in the chromatographic separation and the optimum condition for a particular column resulted in the elution of famciclovir, within approximately 6. 9 min.
Graph : III
Preparation Of Mobile Phase :
Preparation Of Buffer:
2.7218gm of KH 2 PO 4 dissolved in 1000 ml water and final concentration made up to 0.02M.
Preparation Of Ion-Pair Reagent Solution:
468.06 mg of sodium octyl sulphonic acid dissolved in 1000 ml distill water and final concentration made up to 0.002M.
Mobile – Phase:
Taken 1000 ml beaker in that 50 ml of acetonitrile, to this add 468.06 mg of sodium octyl sulphonic acid and 2.7218gm of KH 2 PO 4 added, finally 950 ml distill water added , made up to 1000ml .Finally pH =2.5 adjust with ortho phospheric acid.
Mobile – Phase Ratio:
Acetonitrile –sodium octyl sulphonic acid
(2mM,pH=2.5) KH 2 PO 4 (20mM) (5:95 v/v).
Standard Preparations:
A stock solution of Famciclovir (1000 mg/mL) was prepared by dissolving the powder in the mobile phase. From that 1-5 ml solution taken in five 100 ml volumetric flask and dilute up to 100ml with mobile phase .
Sample Preparation:
20 Intact tablets (VIROVIR) were weighed accurately to obtain the average tablet weight, the tablets were then crushed and triturated in a mortar until a fine powder was obtained. An amount of the powder equivalent to one tablet was weighed accurately and taken in a 100 mL volumetric flask. The powder was dissolved in 25 mL acetonitrile and the solution was diluted to volume with mobile phase: acetonitrile-sodiumoctyl sulphate (2 mM, pH 2.5); KH 2 PO 4 (20 mM) (5:95 v/v).
Dilution (1:10) from this solution was made and the clear solutions were then ready for injection (20 mL was injected).
Assay :
Calculation :
Table– III
Evaluation Of Famciclovir In Pharmaceutical Formulations
|
S. No |
Sample* |
Labelled amount (mg) |
Amount obtained by proposed method |
Percentage recovery + |
|
1. |
T 1 |
250 |
246.819 |
98.72% |
|
2. |
T 2 |
250 |
245.374 |
98.14% |
* Tablets from different companies
+ Average of eight determinations
HPLC Method Validation For Famciclovir:
Linearity: Linearity over the range 1-100 mg/mL of Famciclovir was examined and determined by plotting the peak height response vs. the concentration. The results showed that the method was linear in accordance with Beer’s law over this range and the linearity equation was y = 3.667 x + 1.2310 and regression coefficient was r = 0.9996 (n=6).
Table III A
|
S. No |
Concentration mcg/ml |
Area |
|
1 |
10 |
35.71 |
|
2 |
20 |
71.40 |
|
3 |
30 |
107.10 |
|
4 |
40 |
143.80 |
|
5 |
50 |
184.733 |
Graph IV
Repeatability:
Ten 20 mL injections from a standard solution were injected on to the analytical column and the peak area data obtained were used in assessing system suitability and quality of analysis by calculating the relative standard deviation (RSD=0.094%, n=10) at 30 mg/mL.
Specificity
The specificity of the method was ascertained by analyzing standard drug and sample. The retention time (RT) of Famciclovir was confirmed by comparing the RT with that of the standard fig. 1. The use of placebo like CaCo 3 (calcium carbonate , starch , talc in different concentration was also studied by spiking the standard Famciclovir solution and no interference was observed in the chromatogram.
Accuracy:
Accuracy expresses the closeness of agreement between the value, which is accepted either as conventional true value or and accepted reference value (International Standard, e.g.pharmacopoeal standard) and the value found (mean value) obtained by applying the test procedure a number of times. The results obtained are shown in Table-V from these results, we can conclude that the method was accurate and precise.
Table– IV
|
N |
Cr ( mg/mL) |
The same day |
20 days later |
||||
|
Cm ( mg/mL) |
SD |
RSD (%) |
Cm ( mg/mL) |
SD |
RSD (%) |
||
|
10 |
30 |
30.01 |
0.014 |
0.048 |
29.94 |
0.0042 |
0.072 |
|
10 |
50 |
49.95 |
0.087 |
0.174 |
50.07 |
0.053 |
0.105 |
Cr : True value, Cm : mean concentration. For chromatographic – see text.
Recovery: The recovery of extracted Famciclovir was estimated either by comparing peak heights obtained from extracted aqueous standard and peak heights obtained from spiked standards or more convenient by comparing the slopes of the equations.
In this case the recovery of Famciclovir was 95% (y st = 3.6667 x + 1.2310 and y’ = 3.48365 x’- 0.82477).
LOD: The detection limit (DL) is the lowest concentration of the analyte that can be detected, but not necessarily quantitated, under the stated experimental condition. The absolute limit of detection defined as signal-to-noise of 2 was examined and found to be 3 ng/mL.
LOQ: The Quantitation limit is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions of the method. The limit of quantization (LOQ) was 0.1 mg/mL (20mL) was injected.
Robustnes :
To determine the robustness of the developed method experimental conditions were purposely altered and evaluated. In all the deliberate varied chromatographic conditions (Flow rate -0.9ml/min and 1.1 ml /min, acetonitrile 93% and 97% in the mobile phase, column temperature
30 o to 40 o C). The resolution was more than 2, illustrating the robustness of the method.
Perform experiments by changing conditions slightly such as temperature (±5 o C) buffer pH (± 0.5) ionic strength of buffer, and level of additives to Mobile phase. The method must be robust enough to withstand such slight changes and allow routine analysis of the sample.
Solution Stability :
The solution stability of Famciclovir in the assay method was carried out by leaving both the test solutions of sample and reference standard in tightly capped volumetric flasks at room temperature for two days. The same sample solution were assayed for six hours interval up to the study period. The % RSD of assay of Famciclovir during solution stability experiments was with in 1.0%.
System Suitability Parameters And Recommendations:
Table – V
System suitability parameters and recommendations
|
Parameters |
Recommendations |
Results |
|
Capacity factor ( k ') |
The peak should be well resolved from other peaks and the void volume, generally k ' >2.0 |
The peak well resolved, k ' = 2.61 |
|
Repeatability |
RSD £ 1% for N ³ 5 is desirable |
RSD = 0.94% |
|
Resolution (R s ) |
R s of > 2 between the peak of interest and the closest eluting potential interferent (impurity, excipient, degradation product, internal standard, etc.) |
In NaOH = 2.321, In HCLO 4 = 5.323 |
|
Tailing factor (T) |
T of £ 2. |
T = 1.6 |
|
Theoretical plates (N). |
In general should be > 2000. |
N = 8313 |
Summary :
A reversed phase HPLC method was developed to separate Famciclovir (antiviral agent) from its major related impurities. These agents may participate in a number of interactions including formation of neutral ion-pair and complexation with stationary phase silanols. In optimizing the separation of Famciclovir from its degradation product and plasma, stepwise optimization was carried out. The effect of concentration of acetonitrile (ACN), as the organic modifiers, on the retention of Famciclovir were examined. The effect of the mobile phase and pH on the retention and peak shape of Famciclovir was also examined and its influence on the capacity factor at constant concentration. The results show the best improvements in peak shape at pH 2.5. The method developed is selective, precise, reproducible and accurate and meets the needs of pharmacokinetics studies.
|
Sample Name : FAMCICLOVIR – 50 mcg/ml System : HPLC Detector : UV – VIS Type of Analysis : Percent on Area |
Result Table (Uncal – FAMCICLOVIR – PROJECT-001)
|
Reten. Time [min] |
Area [mV. s) |
Area [%] |
|
|
1 |
1.86 |
3.871 |
2.1 |
|
2 |
6.92 |
180.862 |
97.9 |
|
|
Total |
184.733 |
100.0 |
Fig IV
|
Reten. Time [min] |
Area [mV. s) |
Area [%] |
|
|
1 |
6.84 |
107.102 |
100.00 |
|
|
Total |
107.102 |
100.00 |
Fig V
|
|
Reten. Time [min] |
Area [mV. s) |
Area [%] |
|
1 |
6.86 |
102.503 |
100.00 |
|
|
Total |
102.503 |
100.00 |
Fig VI
|
Sample Name : FAMCICLOVIR – pH 2.5, ACN System : HPLC Detector : UV – VIS Type of Analysis : Percent on Area |
Result Table (Uncal – FAMCICLOVIR – PROJECT-004)
|
|
Reten. Time [min] |
Area [mV. s) |
Area [%] |
|
1 |
6.86 |
102.503 |
100.00 |
|
|
Total |
102.503 |
100.00 |
Fig VII
|
Sample Name : FAMCICLOVIR – ION PAIR 0.002M System : HPLC Detector : UV – VIS Type of Analysis : Percent on Area |
Result Table (Uncal – FAMCICLOVIR – PROJECT-005)
|
|
Reten. Time [min] |
Area [mV. s) |
Area [%] |
|
1 |
6.86 |
102.503 |
100.00 |
|
|
Total |
102.503 |
100.00 |
Fig VIII
|
Sample Name : FAMCICLOVIR – TABLET I System : HPLC Detector : UV – VIS Type of Analysis : Percent on Area |
Result Table (Uncal – FAMCICLOVIR – PROJECT-006)
|
|
Reten. Time [min] |
Area [mV. s) |
Area [%] |
|
1 |
6.86 |
102.503 |
100.00 |
|
|
Total |
102.503 |
100.00 |
Fig IX
|
Sample Name : FAMCICLOVIR – TABLET II System : HPLC Detector : UV – VIS Type of Analysis : Percent on Area |
Result Table (Uncal – FAMCICLOVIR – PROJECT-007)
|
|
Reten. Time [min] |
Area [mV. s) |
Area [%] |
|
1 |
6.86 |
101.903 |
100.00 |
|
|
Total |
101.903 |
100.00 |
Fig X
|
Sample Name : FAMCICLOVIR PERFORMANCE System : HPLC Detector : UV – VIS Type of Analysis : Percent on Area |
Result Table (Uncal – FAMCICLOVIR – PROJECT-008)
|
|
Reten. Time [min] |
Asymmetry [-] |
Efficiency (th.pl) |
|
1 |
6.9 |
1.60 |
8313 |
Fig XII
Stability Indicating Assay For Famciclovir By HPLC
Introduction:
Stability indicating assay 2-3 can be described as a method of analysis by which the parent constituent(s) of the pharmaceutical product can be selectively analyzed. It can be developed either to determine only the active or only the degradation product or the method can be used to separate and determine the parent compound in presence of its impurities and degradation products. The choice of the method depends upon the chemical nature of the drug, the number of impurities, the flexibility of the dosage from and, of course, on practical considerations relating to the availability of resources and instruments.
Currently, chromatographic methods are the methods of first choice for controlling, conformity, purity, stability and strength of the active ingredient. A logical and systematic approach to the development of a stability indicating assay can be carried out initially through a review of internal information present within the laboratory or company or from extern information, from literature sources 22-24 . In addition, biological, toxicological, pharmacological information, physicochemical properties including solubility, partition coefficient, and dissociation constant and spectrophotometric properties should also be reviewed. One method of checking impurities in the active ingredient is to subject it to conditions of stress, such as heat, light and moisture in order to predict probable routes of degradation. Short and long term stability studies of the active ingredient routes of degradation. Short and long term stability studies of the active ingredient and storage conditions should also be examined. On completing development of the method it must be evaluated and therefore validated according to the criteria of specificity, linearity, precision, accuracy, sensitivity and reproducibility. There is no report yet on the development of stability indicating assay method for the drug famciclovir.
This study relates to famciclovir2 - [2 - (2 – amino – 9H – purin – 9 – y1) ethyl] trimethylene diacetate) which is a modified nucleoside that shows strong activity against viruses of the herpes group. It derives from guanine, which is a constituent of purine. In the previous work, a reverse phase ion pair HPLC method developed to separate Famciclovir from its major degradation product, guanine 1 . The objective of the current study was to develop a validated stability indicating assay method for Famciclovir after subjecting the drug to stress conditions.
Experimental
The chromatographic system consisted of Shimadzu LC 20A pump (Touzart, Matignon, France) connected to a Rheoyden 7520 syringe loading sample injector valve fitted with a 20 mL sample loop, a variable wavelength UV detector (UV SPD 20 A) and a Shimadzu chart recorder. The column was a 250 x 4.6 mm, packed with Gemini ODS C18, 5 mm particle size (Phenomenox). Famciclovir was obtained from Cipla Limited, Daman . Famciclovir (VIROVIR) tablets were obtained from FDA.
Results And Discussion
1. Effect Of Temperature :
Tablets of Famciclovir extracted in method phase were subjected to temperature in the range 35-60 0 C. The centrifuge tubes containing analytes were kept in thermostat bath at different temperature. For each tube 20 mL of a particular solution were injected on to the column. A full chromatogram was then obtained and showed no effect and the drug was stable to temperature in the range 35-60 0 C. Fig. XIII shows a chromatogram of Famciclovir at standard temperature (35 0 C). Fig. XIV shows a chromatogram of Famciclovir at standard temperature (60 0 C).
2. Effect Of Light :
In order to test photo degradation, the same tablets were subjected to the effect of UV light and the chromatogram obtained was compared to a reference chromatogram. A full chromatogram was then obtained and showed no effect and the drug was stable. For photo degradation the drug substance in solid state was exposed to UV at 1 lumens for 7 days. Fig XV shows a chromatogram of Famciclovir at UV light.
3. Extraction With 0.5 M Of Naoh Solution :
In assessing the accelerated degradation profile both bulk Famciclovir pharmaceutical tablet (VIROVIR) formulation were extracted with 0.5M sodium hydroxide reflux 30 minutes and neutralize with acid. Full chromatograms were recorded and compared with that extracted in mobile phase composition. As from Fig (XVI). There at some impurities or degradation products related to the drug.
4. Extraction With 1 M Of Naoh Solution :
In assessing the accelerated degradation profile both bulk Famciclovir pharmaceutical tablet (VIROVIR) formulation were extracted with 1 M sodium hydroxide reflux 30 minutes and neutralize with acid. Full chromatograms were recorded and compared with that extracted in mobile phase composition. As from Fig (XVII). There at some impurities or degradation products related to the drug.
5. Extraction With 0.5 M Of Hcio 4 :
In assessing the accelerated degradation profile both bulk Famciclovir pharmaceutical tablet (VIROVIR) formulation were extracted with 0.5 M HCIO 4 reflux 30 minutes and neutralize with base. Full chromatograms were recorded and compared with that extracted in mobile phase composition. As from Fig (XVIII). There at some impurities or degradation products related to the drug.
6. Extraction With 1m Of Hcio 4 :
In assessing the accelerated degradation profile both bulk Famciclovir pharmaceutical tablet (VIROVIR) formulation were extracted with 1M HCIO 4 reflux 30 minutes and neutralize with base. Full chromatograms were recorded and compared with that extracted in mobile phase composition. As from Fig. (XIX). There at some impurities or degradation products related to the drug.
Validation Of HPLC Stability Method:
Linearity:
Linearity over the range 1-100 mg/mL of Famciclovir was examined and determined by plotting the peak height response vs. the concentration. The results showed that the method was linear in accordance with Beer’s law over this range and the linearity equation was y = 3.667 x + 1.2310 and regression coefficient was r = 0.9996 (n=6).
Graph V
Repeatability:
Ten 20 mL injections from a standard solution were injected on to the analytical column and the peak area data obtained were used in assessing system suitability and quality of analysis by calculating the relative standard deviation (RSD=0.094%, n=10) at 30 mg/mL.
Specificity
The specificity of the method was ascertained by analyzing standard drug and sample. The retention time (RT) of Famciclovir was confirmed by comparing the RT with that of the standard fig. XIII. The use of placebo like CaCo 3 (calcium carbonate, starch, talc in different concentration was also studied by spiking the standard Famciclovir solution and no interference was observed in the chromatogram.
Accuracy:
Accuracy expresses the closeness of agreement between the value, which is accepted either as conventional true value or and accepted reference value (International Standard, e.g.pharmacopoeal standard) and the value found (mean value) obtained by applying the test procedure a number of times. The results obtained are shown in Table-VII from these results, we can conclude that the method was accurate and precise.
Table- VI
|
N |
Cr ( mg/mL) |
The same day |
20 days later |
||||
|
Cm ( mg/mL) |
SD |
RSD (%) |
Cm ( mg/mL) |
SD |
RSD (%) |
||
|
10 |
30 |
30.01 |
0.014 |
0.048 |
29.94 |
0.004 |
0.072 |
|
10 |
50 |
49.95 |
0.087 |
0.174 |
50.07 |
0.053 |
0.105 |
Cr : True value, Cm : mean concentration.
Recovery:
The recovery of extracted Famciclovir was estimated either by comparing peak heights obtained from extracted aqueous standard and peak heights obtained from spiked standards or more convenient by comparing the slopes of the equations.
In this case the recovery of Famciclovir was 95% (y st = 3.6667 x + 1.2310 and y’ = 3.48365 x’- 0.82477).
Robustness
The robustness was tested by introducing small changes in the, mobile phase composition. Mobile phase having different compositions of ACN: water (95:5 ± 0.5 v/v) were tried and chromatograms obtained. Also the operating temperature and relative humidity was varied in the range of ± 0.5%. Robustness of the method was done at two different concentration levels of 20 and 50 mcg/ ml.
Solution Stability
The solution stability of Famciclovir in the related substance method was carried out by leaving sample solution in tightly capped volumetric flask at room temperature for two days. Content of degradation products were checked for six hours. Interval upto the study period No significant change was observed in the content during solution stability experiments upto the study period. Hence Famciclovir sample solution are stable for at least 48 hours in the developed method.
LOD:
The detection limit (DL) is the lowest concentration of the analyte that can be detected, but not necessarily quantitated, under the stated experimental condition. The absolute limit of detection defined as signal-to-noise of 2 was examined and found to be 3 ng/ml.
LOQ: The Quantitation limit is the lowest concentration of analyte in a sample that can be determined with acceptable precision and accuracy under the stated experimental conditions of the method. The limit of quantization (LOQ) was 0.1 mg/mL (20mL) was injected.
System suitability parameters and recommendations
|
Parameters |
Recommendations |
Results |
|
Capacity factor ( k ') |
The peak should be well resolved from other peaks and the void volume, generally k ' >2.0 |
The peak well resolved, k ' = 2.61 |
|
Repeatability |
RSD £ 1% for N ³ 5 is desirable |
RSD = 0.94% |
|
Resolution (R s ) |
R s of > 2 between the peak of interest and the closest eluting potential interferent (impurity, excipient, degradation product, internal standard, etc.) |
In NaOH = 2.321, In HCLO 4 =5.323 |
|
Tailing factor (T) |
T of £ 2. |
T = 1.6 |
|
Theoretical plates (N). |
In general should be > 2000. |
N = 8313 |
Summary :
The influence of stability of Famciclovir in severe conditions was tested (acid and alkaline environment) and the Famciclovir impurities were evaluated using ion-pairing HPLC method and mobile phase H 2 O : ACN
5: 95% (v/v) counter-ion C 8 H 17 SO 4 Na 0.002 M, pH 2.5, KH 2 PO 4 0.02 M, flow rate 2 mL/min, UV detection 314 nm. The method was precise and linear (R>0.9996, n=6) the range of 1-100 mg/mL .
|
Sample Name : FAMCICLOVIR – 50 mcg/ml System : HPLC Detector : UV – VIS Type of Analysis : Percent on Area |
Result Table (Uncal – FAMCICLOVIR – PROJECT-009)
|
|
Reten . Time [min] |
Area [mV. s) |
Area [%] |
|
1 |
1.86 |
3.871 |
2.1 |
|
2 |
6.92 |
180.862 |
97.9 |
|
|
Total |
184.733 |
100.0 |
Fig XII
|
Sample Name : FAMCICLOVIR – STABILITY AT 35 0 C System : HPLC Detector : UV – VIS Type of Analysis : Percent on Area |
Result Table (Uncal – FAMCICLOVIR – PROJECT-010)
|
|
Reten . Time [min] |
Area [mV. s) |
Area [%] |
|
1 |
6.86 |
107.102 |
100.00 |
|
|
Total |
107.102 |
100.00 |
Fig XIII
|
Sample Name : FAMCICLOVIR – STABILITY AT 60 0 C System : HPLC Detector : UV – VIS Type of Analysis : Percent on Area |
Result Table (Uncal – FAMCICLOVIR – PROJECT-011)
|
|
Reten . Time [min] |
Area [mV. s) |
Area [%] |
|
1 |
6.86 |
107.102 |
100.00 |
|
|
Total |
107.102 |
100.00 |
Fig XIV
|
Sample Name : FAMCICLOVIR – UV LIGHT System : HPLC Detector : UV – VIS Type of Analysis : Percent on Area |
Result Table (Uncal – FAMCICLOVIR – PROJECT-012)
|
|
Reten . Time [min] |
Area [mV. s) |
Area [%] |
|
1 |
6.86 |
107.102 |
100.00 |
|
|
Total |
107.102 |
100.00 |
Fig XV
|
Sample Name : FAMCICLOVIR – STABILITY 0.5 M NaOH System : HPLC Detector : UV – VIS Type of Analysis : Percent on Area |
Result Table (Uncal – FAMCICLOVIR – PROJECT-013)
|
|
Reten . Time [min] |
Area [mV. s) |
Area [%] |
|
1 |
6.9 |
175.049 |
94.4 |
|
2 |
7.2 |
10.288 |
5.6 |
|
|
Total |
185.337 |
100.0 |
Fig XVI
|
Sample Name : FAMCICLOVIR – STABILITY 1 M NaOH System : HPLC Detector : UV – VIS Type of Analysis : Percent on Area |
Result Table (Uncal – FAMCICLOVIR – PROJECT-014)
|
|
Reten . Time [min] |
Area [mV. s) |
Area [%] |
|
1 |
6.7 |
4.26 |
2.3 |
|
2 |
6.82 |
168.17 |
90.7 |
|
3 |
7.2 |
12.979 |
7.0 |
|
|
Total |
185.409 |
100.0 |
Fig XVII
|
Sample Name : FAMCICLOVIR – STABILITY 0.5 M HCLO 4 System : HPLC Detector : UV – VIS Type of Analysis : Percent on Area |
Result Table (Uncal – FAMCICLOVIR – PROJECT-015)
|
|
Reten . Time [min] |
Area [mV. s) |
Area [%] |
|
1 |
2.8 |
30.535 |
16.6 |
|
2 |
3.31 |
4.858 |
2.6 |
|
3 |
6.91 |
148.719 |
80.8 |
|
|
Total |
184.112 |
100.0 |
Fig XVIII
|
Sample Name : FAMCICLOVIR – STABILITY 1 M HCLO 4 System : HPLC Detector : UV – VIS Type of Analysis : Percent on Area |
Result Table (Uncal – FAMCICLOVIR – PROJECT-016)
|
|
Reten . Time [min] |
Area [mV. s) |
Area [%] |
|
1 |
2.6 |
6.502 |
3.5 |
|
2 |
2.8 |
51.446 |
28.0 |
|
3 |
3.31 |
14.844 |
8.08 |
|
4 |
6.92 |
110.782 |
60.3 |
|
|
Total |
183.574 |
100.0 |
Fig XIX
Bibliography
1. The use of Antibiotics, A clinical Review of Antibacterial, Antifungal and Antiviral Drugs, 5th Edn., p. 1542 (2001).
2. David .J. Mazzo (Ed.), International Stability Testing.
3. Jenst. Carstensen C.T. Rhodes, Marcel, Dekker, The Drug Stability, 17, 329.
4. M.J.O’Neil (Ed.), The Merk Index: An Encyclopedia of Chemicals, Drugs and Biologicals, Merk & Co., Inc., 13th Edn., p. 695 (2001).
5. R. Harnden et al., J. Med. Chem., 32 , 1738 (1989).
6. J.R. McMeekin et al. , Anal. Proc., 29 , 178 (1992).
7. R.A. Vere Hodge et al., Antivir. Chem. Chemother ., 4 , 67 (1993).
8. R. Circelli et al., Antivir. Res., 29 , 141 (1996).
9. G. Land and A.Bye, J. Chromotogr ., 224 , 51 (1981)
10. R.L. Smith and D.D. Walker, J. Chromatogr., 343 , 203 (1985)
11. K.J Swart, H.K.L. Hundt and A.M. Croenawald, J. Chromatogr. , A 663 , 65 (1994).
12. M.A. Campanero, B.Sabada, E. Garcioquetglas and J.R. Azanza.
J.Chromatogr ., B, 706, 311 (1998).
13. Gloor R. and F.L. Johnson, J. Chromatogr., Sci., 15, 413 (1977).
14. G. Land, N. Shortman and G. Ridout, J. Chromatographia, 19, 310 (1984).
15. R. Causon. J. Chromatogr., B. 689, 175 (1997).
16. S.M. Tadepalli and R.P. Quinn, J. Pharm, Biomed. Anal., 15, 157 (1996).
17. C.M. Riley, J.M. Ault (Jr.) and N.E. Klutman., J. Chromatogr, 531, 295 (1994).
18. D.S. Aston, C. Beddell, A.D. Ray and K. Valko, J. Chromatogr. A. 707, 367 (1995).
19. G. Stark, J.P. Fawcett and I.G. Tucker, J. Pharm., 258, 637 (1997).
20. J.Y. Ortholand, Chem.. Eng . , 6, 47 (1998).
21. List of Guidance Documents, CDER. http: // www. fda. Gov / cder/guidance/index.htm.
22. Useful websites:
About Author:
K.V.Subrahmanyam
Corresponding Adress:
s/o Gangadhara Rao, Mopidevi (p.o), Krishna , A.P., India -521125, +91 9440850969
E-Mail : subhashguy@yahoo.co.in
Dr.N.Gopal
c/o Nanda college of pharmacy ,Erode,Tamilnadu, +91 9440850969
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