FIELD OF THE INVENTION
The present invention pertains to the field of pharmaceutical analysis. More specifically it pertains to quantification of gabapentin without any derivatization in bulk, pharmaceutical formulations and human urine samples.
BACKGROUND OF THE INVENTION
Gabapentin, 1-(amino methyl)-cyclohexane acetic acid is a structural analogue of the neurotransmitter g-aminobutyric acid (GABA) (Fig. 1) with a molecular formula of C9H17NO2 and a molecular weight of 171.24. It is a white crystalline solid, which is highly charged at physiological pH, existing as a zwitterion with a pKal of 3.7 and a pKa2 of 10.7. It is freely soluble in water in both basic and acidic aqueous solutions.
(STRUCTURE REMOVED)
It is an antiepileptic drug, which has been found to have pain-relieving properties. Gabapentin is indicated for the management of post-herpetic neuralgia in adults. It is reported as adjunctive therapy in the treatment of partial seizures with and without secondary generalization in patients over 12 years of age with epilepsy and in the treatment of partial seizures in pediatric patient's age 3-12 years. Additionally, it is indicated to have anti-anxiety activity as also beneficial properties in treating neurodegenerative diseases like Alzheimer's.
Significance of Gabapentin Estimation
In humans, gabapentin is not metabolized and it is eliminated unchanged in the urine and any unabsorbed drug is excreted in the faeces. Thus, analyzing gabapentin in various biological fluids is an important criterion for the determination of the physiological performance of a drug because of the following facts:
1. Wide individual variation in the rate of clearance of this drug.
2. Always a need to perform compliance testing, ascertain toxicity and elucidate possible clinical interactions.
So, the therapeutic monitoring of gabapentin is highly desirable as it can serve as an excellent tool to ensure compliance in administering chemotherapy with the actual prescribed dosage and achievement of the effective serum concentration levels.
Methods of Gabapentin Estimation in prior art and their limitations
Numerous analytical methods have been reported in the literature for the determination of gabapentin in pure form, human serum and pharmaceutical formulation but suffer from one limitation or the other mainly requirement of dedicated and expensive instrumentation or cumbersome derivatization.
Expensive instrumentation
Existing methods for gabapentin estimation are based on gas chromatography (Kushnir et a/., 1999; Wolf et al, 1996), fluorimetry (Belal et al, 2002), capillary electrophoresis (Garcia etal, 1995; Lin et al, 2004) and gas chromatography - mass spectrophotometry (Borrey et al, 2005) following derivatization.
Cumbersome derivatization
Most of the HPLC assay procedures for the determination of gabapentin have been published using different derivatizing agents but they suffer from limitations such as a lengthy run time or using special reaction conditions which may not be suitable for routine analysis.
These are based on the same approach, involving a simple automated approach of O-opthaldehyde (OPA) derivatization followed by HPLC separation in acidic mobile
phases and fluorimetric detection. However, this method suffers from the limitation that
the derived product - fluorescent OPA - drug derivative is highly unstable and should be
injected immediately after preparation. Therefore, the method is difficult to apply for
routine studies especially when automated instrumentation is not available (Gauthier
and Gupta,2002; Vermeij eta/., 2004).
Derivatization with potassium iodate and potassium iodide requires more reagents and
it is detected at higher UV wavelength but it is a good method for analysis (Gujral and
Haque, 2009).
Derivatization with phenyl - isothiocyanate (PITC) is simple, but this reagent degrades
in contact with water and the reaction medium should be completely free from water
before the addition of reagent (Zhu and Neirinck, 2002).
Derivatization with 4 - chloro - 7 nitrobenzofurazon as a labeling reagent, the adduct
should be extracted and detected by spectrofluorimetry (Bahrami and Mohammadi,
2006). The reaction time for gabapentin and 9 - fluorenyl methyl chloroformate is not too
long (about 10 min), but a column temperature of 60°C is needed for separation of the
adduct (Bahrami and Kiani, 2006).
In one of the methods, spectrofluorimetric determination of gabapentin was reported after
derivatization with fluorescamine (Belal et aL, 2002).
In another case, colorimetric determination of gabapentin was studied based on the
reaction with vanillin and ninhydrin (Abdellatef and Khalil, 2003).
Thus, in all the methods of analysis reported in prior art, the major limitation was derivatization and requirement of special detector systems. They could not be performed on simple and easily available detector systems such as UV detectors. The derivatization condition was time consuming and the stability of the reaction products depended on experimental conditions such as pH, temperature and reaction time. The gas chromatography methods reported so far required complex sample preparation involving double derivatization of the drugs to improve the volatility and avoid column interactions. Comparing the capillary electrophoresis and HPLC methods, the later is more precise, reproducible, and sensitive than the former, although the many advantages of capillary electrophoresis, such as smaller injection volume, simplicity and wide
applicability, should be taken into account. Fluorometric methods are less accurate and less specific than HPLC.
The process of the present invention has been able to overcome all these limitations as it permits the easy estimation of Gabapentin directly, eliminating the need for cumbersome derivatization and enabling use of easily available instrumentation in form of UV detector system attached to HPLC. The present invention discloses a rapid, sensitive, accurate and precise HPLC method for determination of gabapentin without derivatization in bulk, pharmaceutical formulations and human urine samples.
BIBLIOGRAPHY
1. Abdellatef HE and Khalil HM (2003) JPharm BiomedAnal 31: 209 - 214.
2. Bahrami G and Mohammadi B (2006) J Chromatogr B 837: 24 - 28.
3. Belal F, Abdine H, Al - Majed A and Khalil NY (2002) J Pharm BiomedAnal 27: 253 - 260.
4. Borrey DCR, Godderis KO, Engerlrelst VIL, Bernard DR and Langlois MR (2005) Clin Chim Acta 354: 147-151.
5. Garcia LL, Shihabi ZK and Oles K (1995) J Chromatogr B 669: 157 - 162.
6. Gauthier JD and Gupta R (2002) Clin Chem 48: 2259 - 2261.
7. Gujral RS and Haque SM (2009) Int JBiomed Sci 5: 100 - 106.
8. Kushnir MM, Crossett J, Brown PI and Urray FM (1999) J Anal Toxicol 23: 1 - 6.
9. Lin FM, Lin HS, Kou HS, Wu SM, Chen SH and Wu HL (2004) Anal Chim Act 523: 9-14.
10. Vermeij TAC and Edelborek PM (2004) J Chromatogr B 810:297- 303.
11. Wolf CE, Saady JJ and Poklis A (1996) J Anal Toxicol 20: 498 - 501.
12. Zhu Z and Neirinck L (2002) J Chromatogr B119: 307- 312.
OBJECTS OF THE INVENTION
The present invention has the following objects: 1. To disclose a novel, rapid, sensitive, accurate and precise HPLC method for determination of gabapentin without derivatization
2. To disclose an improved process for gabapentin detection which overcomes the drawbacks of previously reported methods mainly cumbersome derivatization and need for dedicated and expensive instrumentation.
3. To disclose a UV detection method for gabapentin determination in bulk, pharmaceutical formulations and human urine samples in vitro.
SUMMARY OF THE INVENTION
The present invention discloses a novel process for determination of gabapentin without derivatization, in bulk, pharmaceutical formulations and human urine samples by HPLC using UV detector.
BRIEF DESCRIPTION OF THE DRAWINGS
NIL - No drawings attached
DETAILED DESCRIPTION OF THE INVENTION
The present invention describes a rapid, sensitive, accurate and precise HPLC method for determination of gabapentin without derivatization in bulk, pharmaceutical formulations and human urine samples.
NOVELTY ASPECT OF INVENTION
The present invention discloses a novel process for determination of gabapentin without derivatization using an HPLC system with UV detector, which has not been reported in prior art to the best of inventor's knowledge.
INVENTIVE STEP
The inventive step of novel process of HPLC is the optimization of chromatographic conditions which permit gabapentin estimation directly, without need for derivatization and by use of easily available UV detector, thus permitting faster and more convenient analysis. This was achieved by monitoring various columns systems and trying different ratios of mixtures of methanol - acetonitrile - potassium dihydrogen orthophosphate as mobile phase in the present invention.
Excellent chromatographic specificity with no interference from dosage form excipients was observed and moreover, a suitable retention time for gabapentin was achieved in the present invention. So the performance of the novel method in terms of accuracy and relative standard deviation (RSD) values was much better and it does not require any complex system for dissolution of samples, dissolution medium for the analysis. The proposed method does not require any laborious clean up procedure before measurement and can be used for low purity (94.56 % and 88.65%) gabapentin. Therefore, the present method of invention is simple, sensitive, rapid and can compete with other existing HPLC method for the determination of gabapentin in its pharmaceutical formulations and for in vitro determination in human urine samples.
INDUSTRIAL APPLICATION
The present method is simple, fast and cheap. It can be used by API (active pharmaceutical ingredient) manufacturers for monitoring quality of the batch, formulation manufacturers for monitoring quality/stability and other aspects of formulations and finally by clinical research organizations who are monitoring the levels of the drug for various aspects of R&D relating to safety, efficacy etc.
NOVEL PROCESS FOR DIRECT ESTIMATION OF GABAPENTIN
A simple and reliable HPLC method with UV detection has been developed in present invention and validated for the determination of gabapentin in bulk, pharmaceutical formulations and human urine samples. Here HPLC stands for high performance liquid chromatography. It is a liquid chromatography which involves the separation of the compounds on the basis of their polarity. It is used to analyze, identify, purify & quantify the compounds. In HPLC, analyte is forced through a column which is usually a tube packed with small round particles with a certain surface chemistry (stationary phase) by pumping a liquid (mobile phase) at high pressure through the column. The amount of retardation depends on the nature of the analyte, stationary phase and mobile phase composition. The time at which a specific analyte elutes (comes out of the end of the column) is called the retention time and is considered a reasonably unique identifying characteristic of a given analyte. The use of pressure increases the linear velocity (speed)
giving the components less time to diffuse within the column, leading to improved resolution in the resulting chromatogram.
UV (ultra violet) detector is the component of HPLC which is positioned immediately posterior to the stationary phase in order to detect the compounds as they elute from the column and subsequently signals a peak on the chromatogram. It functions on the principle that many compounds tend to absorb light in the wavelength range of 180 to 350 nm. It has the best combination of sensitivity, linearity, versatility and reliability of all the liquid chromatography detectors so far developed.
TECHNICAL DETAILS OF THE NOVEL PROCESS
These are described below: Preparation of solutions for HPLC
1. Stock standard solution of gabapentin: [Standard USP Gabapentin Lot No GOE005 (0.999 mg/mg)] was prepared by dissolving an appropriate amount of the compound in mobile phase having final concentration of 5 mg/ ml. Standard solutions of gabapentin (0.75, 2.0 and 3.2 mg/ml) were prepared by subsequent dilution.
2. Mobile phase: The mobile phase consisted of methanol - acetonitrile - potassium dihydrogen phosphate in the ratio of 25:10:65 (v/v) respectively [Here phosphate buffer of 0.028 M potassium dihydrogen orthophosphate (KH2PO4) (2.50 gm in 650 ml distilled water) was prepared having pH of 5.2 which is adjusted by adding 10 % sodium hydroxide (NaOH) (10 gm in 100 ml distilled water)].
Chromatographic conditions
Chromatographic conditions were optimized by monitoring various columns and mobile systems. Silica columns such as a u Bondapak column with different mobile phases did not give a suitable peak shape for analysis as compared to waters Cig column. Different ratios of mixtures of methanol - acetonitrile - potassium dihydrogen orthophosphate as mobile phase were evaluated. The present invention discloses a chromatographic separation which was achieved using Waters Cis 5 nm column (150 mm x 4.6 mm) and the isocratic mobile phase consisted of methanol - acetonitrile - potassium dihydrogen phosphate (pH 5.2; 0.028 M) (25:10:65, v/v). Mobile phase was pumped at a flow rate of
1.0 ml/ min. It was prepared daily and degassed by passing through a 0.45 urn filter and ultrasonication for 10 min. All separations were performed at room temperature with detection at 210 nm. Under the chromatographic conditions described, gabapentin was well resolved and eluted at about 2.36 min and the total run time was within 30 min. Good baseline resolution and peak shape was observed in the typical chromatogram as shown in Fig. 2 obtained from the standard solution of gabapentin, assay preparation of Gabacom tablet and a test solution from dissolution medium of Gabacom tablet.
(TABLE REMOVED)
Fig. 2 HPLC chromatograms of gabapentin (2 mg/ ml).
Procedure for Gabapentin Determination
Aliquots of stock solution (5 mg/ml) were transferred in to a 10 ml volumetric flask and volumes were completed to the mark with the mobile phase to produce solutions in the concentration range 0.1-3.8 mg/ml. Twenty micro liters of the solution was injected into the HPLC system and eluents were detected by the UV detector with the wavelength of 210 nm. The signals emerging from the detector were integrated as peak area and a calibration graph of peak area against the concentration of gabapentin was plotted. The regression equation was also derived from standard graph of gabapentin as presented in the Fig. 3 and Table 1.
Procedure for the Determination of Gabapentin in Bulk, Pharmaceutical Formulation
The content of one gabacom tablet was combined, weighed and an amount of powder equivalent to about 800 mg gabapentin was accurately weighed, transferred to a 100 ml volumetric flask, made up to volume with distilled water and placed in an ultrasonic bath for 15 min. After passing through a 0.45 um Millipore filter, the solution was diluted with water to obtain a concentration of about 8 mg/ml. The drug concentrations of seven replicates were determined by HPLC using the calibration curve.
Procedure for the Determination of Gabapentin in Human Urine Samples
Aliquot volumes of human urine samples were transferred into small separating funnels. Then 5 ml of carbonate buffer having pH of 9.4 (prepared by dissolving 26.5 gm sodium carbonate and 21 gm sodium bicarbonate in 500 ml distilled water) was added to these
(FIQURE REMOVED)
Fig. 3 Linear regression plot of the proposed method.
Table 1 Optical characteristics and statistical data of the regression equation for the
proposed method.
(TABLE REMOVED)
funnels and mixed well the solution. The solution was then extracted with 3 * 5 ml of diethyl ether. The ether extract was collected and evaporated. The residue was dissolved in 5 ml of distilled water and above general procedure was then followed. The nominal content of gabapentin was determined from the corresponding regression equation.
Procedure for the Determination of Gabapentin by Reference Method
Aliquots of stock solution (14 mg/ml) were transferred into a set of 10 ml volumetric flasks and volumes were completed to the mark with diluents (dissolved 2.32 gm monobasic ammonium phosphate in 1000 ml of water and pH of 2.0 was adjusted with phosphoric acid) to produce solutions in the concentration range of 1000-10000 ug/ ml. Calibration curve were constructed by plotting peak area against the final concentration gabapentin. For reference method the separation was achieved by using 4.6 mm * 25 cm column that contains packing LI. The isocratic mobile phase was pumped at a flow - rate of 1.0 ml/min consisted of acetonitrile - buffer solution (dissolved 0.58 gm of monobasic ammonium phosphate and 1.83 gm of sodium perchlorate in 1000 ml of water and pH of solution was adjusted to 1.8 with perchloric acid).
Validation of various parameters for novel process of HPLC
The validation characteristics included solution stability, specificity, selectivity, linearity, accuracy and precision. Robustness and ruggedness testing was also conducted to evaluate the effect of minor changes to the chromatographic system and to establish appropriate system suitability parameters. Validation acceptance criteria were met in all cases as described below:
Solution stability
The stability of the reference gabapentin and quality control sample solutions at room temperature was ascertained from the respective HPLC peak area. The peak area was obtained at 2.36 min time with a UV detector at wavelength of 210 ran (2.000 AUFS). It was observed that there was no change in peak area of the solutions when these were kept below 5°C for 15 days.
Specificity and selectivity
The specificity and selectivity of the present method was ascertained by analyzing standard gabapentin in the presence of excipients such as sodium stearyl fumarate, magnesium stearate, starch, lactose and talc. It was observed that these excipients did not interfere with the present method of invention.
Linearity
The linearity of the method was ascertained by analysing gabapentin at nine concentration levels 0.1 - 3.8 mg/ml. Each concentration was independently analyzed five times and under the optimum experimental conditions, the peak area - concentration plot for present method was found to be rectilinear. Linear regression analysis of calibration data gave the regression equations with correlation coefficients close to unity cited in Table 1.
Precision and Accuracy
The precision and accuracy of the method was evaluated within the linear range based on the analysis of gabapentin in reference standard samples at 0.75,2.0 and 3.2 mg/ ml. Five
independent analysis were performed at each concentration level within one day (intra day precision) as well as for five consecutive days (inter day precision). The within day precision assays were carried out through replicate analysis (n = 5) of gabapentin corresponding to 0.75, 2.0 and 3.2 mg/ ml for the proposed method. The interday precision was also evaluated through replicate analysis of the pure drug samples for five consecutive days at the same concentration levels as used in the within day precision. The precision results of these assays reported in Table 2 showed that RSD values for within day precision was lower than 0.116 % and RSD values for interday precision was lower than 0.487 % which were quite satisfactory.
Table 2 Test of precision of the proposed method.
(TABLE REMOVED)
The accuracy of the present method was ascertained by recovery studies using the standard addition method. For this, 0.25 (or 0.70, 1.15, 1.75, 2.20, 2.70 and 3.0 mg/ ml) of reference gabapentin solution (5 mg/ml) was transferred into a 50 ml volumetric flask followed by 0.25 mg/ ml of sample solution (5 mg/ ml) and the volume was made up to the mark with the mobile phase. The total amount was determined by the present procedure. The results presented in Table 3 summarize that RSD of the present method was lower than 0.116 ± 1.884 % which agreed well within the acceptable limits of ± 2%.
Table 3 Determination of Gabapentin in pharmaceutical preparation by
standard addition method.
(TABLE REMOVED)
Robustness and Ruggedness of novel method
For the evaluation of ruggedness of the present method, the contents of gabapentin at 3.5 mg/ ml were assayed following the recommended procedure using Shimadzu LC 2010 CHT Auto sampler and Waters 2487 HPLC systems. The recoveries ± RSD resulting from the Shimadzu LC 2010 CHT (99.86 ± 0.669) and Waters 2487 (99.16 ± 0.471) were compared. The robustness of the method relative to operational parameters like mobile phase, ± 2.5 %, buffer pH, 5.2 ± 0.01 %, retention time, 20 ± 2 Min was checked and investigated. The robustness of the method was assessed by analyzing the active gabapentin in Gabacom tablet and the quality control sample solution containing 2.87 mg/ ml of the drug was also assayed. The percent recovery ± RSD of the method (100.12 ± 0.919) was found to be appreciable, indicating the ruggedness and robustness of present method as compared with reported method.
The proposed method for determination of gabapentin in its pharmaceutical formulation was compared with those obtained by the reference method and it was also validated with low purity (94.56 % and 88.65%) gabapentin with reference method as presented in
Table 4. In this case, the accuracy and RSD value of present method is better as compared to reported methods.
Table 4 Determination of Gabapentin by proposed method and reference method.
(TABLE REMOVED)
It was further extended to the in vitro determination of gabapentin in human urine samples in the proposed linearity range. The results of analysis summarized in Table 5 were satisfactorily accurate and precise. Therefore the novel process of the present invention was found to be accurate for in vitro determination of gabapentin in human urine samples.
Table 5 Determination of Gabapentin in human urine samples.
(TABLE REMOVED)
In the above detailed description of the invention, method of optimizing chromatographic conditions and validating statistical parameters has been disclosed. It is to be understood that this invention is not limited to particular embodiments described as such and these may of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

WE CLAIM
1. A novel process for quantification of gabapentin in bulk, pharmaceuticals
formulations and human urine samples based on HPLC wherein the same
comprises direct UV based estimation of the compound without any
derivatization.
2. The process as claimed in claim 1, wherein a chromatographic separation is
achieved using a reverse phase column such as Waters C18, 5 urn column (150
mm x 4.6 mm).
3. The process as claimed in claim 1, wherein the mobile phase used for isocratic
separation is a mixture of methanol - acetonitrile - potassium dihydrogen
orthophosphate (25:10:65, v/v).
4. The process as claimed in claim 3, wherein molarity of the isocratic mobile phase
is optimally 0.028 M.
5. The process as claimed in claim 1 and 3, wherein pH of the isocratic mobile phase
is optimally 5.2.
6. The process as claimed in claim 1 and 3, wherein flow rate of the isocratic mobile
phase is optimally 1.0 ml/ min.
7. The process as claimed in claim 1 and 3 wherein the reference solutions of
gabapentin are prepared in the concentration range of 0.1-3.8 mg/ml using
isocratic mobile phase.
8. The process as claimed in claim 1, wherein sample volume for HPLC is optimally
20 ul and UV detection wavelength is 210 nm.
9. A process for the detection of gabapentin without derivatization in bulk,
pharmaceuticals formulations and human urine samples as described herein with
reference to the detailed description.