Field of the invention:
The present invention relates to the field of pharmaceutical analysis. More particularly it relates
to a novel process for determining the concentration of a drug viz. Pregabalin in bulk,
pharmaceuticals formulations and human urine samples directly, without need for any
derivatization or modification.
Background of the invention:
About Pregabalin: Pregabalin (PGB), (S)-3-amino methyl hexanoic acid is a structural analogue
of Y-amino butyric acid (GABA). It is a white crystalline solid which is soluble in water as well
as in aqueous solutions like acid and base. It is an anticonvulsant drug used for neuropathic pain
and as an adjunct therapy for partial seizures with or without secondary generalization in adults.
Structure of pregabalin:
Molecular Weight: 159.22608 [g/mol]
Molecular Formula: C98H17NO2
Pregabalin was initially developed by medicinal chemist Richard Bruce Silverman at
Northwestern University in the United States. The drug was approved in the European Union in
2004. Pregabalin received U.S. Food and Drug Administration (FDA) approval for use in
treating epilepsy, diabetic neuropathy pain and post-herpetic neuralgia in December 2004 and
reached the U.S. market in 2005. It was designed as a more potent successor to gabapentin.
Pregabalin is marketed by Pfizer under the trade name Lyrica. Recent studies have shown that
pregabalin is effective in treating chronic pain in disorders such as fibromyalgia and spinal cord
injury. In June 2007, pregabalin became the first medication approved by the U.S. Food and
Drug Administration specifically for the treatment of fibromyalgia.
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Significance of the present invention: Pregabalin manufacture as an API (Active
Pharmaceutical Ingredient) or as formulation involves repeated estimations of the same for
quality control. Additionally, clinical studies relating to various aspects of R&D of this drug
necessitate regular estimations in clinical samples e.g. urine, blood serum/plasma. Existing
methods are tedious and require cumbersome instrumentation while the present process is much
faster and can be performed without need of expensive instrumentation.
Prior art related to determination of pregabalin:
There is no reported method for the analysis of pregabalin till now and no existing patent for
determination of pregabalin is available. A thorough literature search has revealed that only a
few analytical methods are available for determination of pregabalin in bulk drugs and
pharmaceutical formulations which are discussed below.
Armagan Onal and Olcay Sagirli (Science direct, Spectrochimica Acta Part A: Molecular and
Bimolecular Spectroscopy, Volume 72, Issue 1, February 2009, Pages 68-71) describe
spectrofluorimetric and spectrophotometric methods for the determination of the y-amino-nbutyric
acid derivative pregabalin (PGB) in bulk drug and capsule. Pregabalin, as a primary
amine compound reacts with 7-chloro-4-nitrobenzofurazon and the reaction product was
measured on spectrofluorimetric and spectrophotometric devices. The mean recovery for the
commercial capsules was 99.93% and 99.96% for spectrophotometric and spectrofluorimetric
study respectively. However, in the present invention the determination of pregabalin is done by
HPLC method (High performance liquid chromatography) in bulk pharmaceutical formulations
as well as in human urine samples without derivatization step and complete process requires less
then 10 minutes with retention time of only 4.632 minutes.
T.A.C. Vermeij and P.M. Edelbroek (Science Direct, J. Chromatography B, Volume 810,
2004, Pages 297 - 303) describe a simultaneous high-performance liquid chromatographic
analysis of pregabalin, gabapentin and vigabatrin in human serum by precolumn derivatization
with o-phtaldialdehyde and fluorescence detection.
Vikas V. Vaidyal , Santosh M. Yetal, Shikha M. N. Roy, Noel A. Gomes and Santosh S.
Joshi (Chromatographia, Volume 66, Numbers 11-12/ December, 2007 Pages 925-928) describe
a rapid, sensitive and specific method to quantify pregabalin in human plasma using metaxalone
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and analysis was done by high-performance liquid chromatography coupled to electrospray
tandem mass spectrometry (LC-MS-MS) in which derivatization step was needed.
Chen, Xiaohui., Zhang, Daolin, Deng, Jie, Fu, Xiaotai (Journal of Chromatographic Science,
Volume 46, Number 1, January 2008 , pp. 42-44 (3)) describe a process for determination of
optical impurity of pregabalin by HPLC with Pre-Column Chiral Derivatization.
All the above processes disclose use of HPLC but with the following limitations viz.
a. Use of expensive detector systems: e.g. fluorescent detector or LC-MS.
b. Need for derivatization: All the disclosed methods in prior art require derivatization of the
compound for analysis.
In contrast, the present invention does not require any derivatization thus reducing time and cost
of the analysis. Secondly, the detector system is simply a UV detector, which is one of the most
commonly available and cheapest detectors available in most analytical labs.
The method described in the present invention is simple, efficient and selective for the analysis
of the PGB in bulk, pharmaceutical formulations and human urine samples because of the
following reasons:
1. It does not require any derivatization step.
2. It utilizes UV detector system which is easily available
3. The time required of the completion of process is approximately 10 minutes.
OBJECTS OF INVENTION:
The invention has the following objects:
1. To provide a novel process for pregabalin determination in a bulk, pharmaceutical
formulations and human urine samples.
2. To provide a novel HPLC method for determining pregabalin in bulk, pharmaceutical
formulations and human urine samples without derivatization with other reagents.
3. To provide a method for determining pregabalin in bulk, pharmaceutical formulations
and human urine samples in a short time period.
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4. To provide a novel process for pregabalin determination in which the equipment used are
HPLC unit with UV detection which is readily available in most analytical and
pharmaceutical laboratories.
SUMMARY OF INVENTION:
The invention relates to a novel HPLC process for pregabalin determination in bulkj
pharmaceutical formulations and human urine samples directly, without need for any
derivatization, prior separation or purification. Also, the method utilizes a simple, readily
available UV detector and does not require any specialized detector systems. The process
described in the present invention is simple, efficient and selective/specific.
DETAILED DESCRIPTION OF THE INVENTION:
The present invention relates to a novel HPLC process for pregabalin determination in bulk,
pharmaceutical formulations as well as in human urine samples which is simpler than other
existing methods because it does not require any derivatization step and can be performed
directly on the samples without need for prior separation or purification. Also, it is rapid, having
a run time of less than 10 minutes and can be performed using easily available UV detector, thus
avoiding the need for expensive and dedicated detector systems such as LC-MS or Flurescence
Detector.
NOVELTY ASPECT OF INVENTION: Novelty aspect of the invention lies in disclosing a
new HPLC process for pregabalin determination in which prior separation, purification or
derivatization is not required before analysis is not required.
INVENTIVE STEP: The inventive step of the invention lies in developing a HPLC method
which does not require derivatization steps for detection and enables quantification to be carried
out using a simple UV detector system, which is easily available in most analytical labs. The
same has been achived by development and use of an appropriate mobile phase comprising of a
mixture of methanol - acetonitrile - 0.02 M di - Potassium Hydrogen Orthophosphate (pH -
7.00) (3:1:16, v/v/v) and standardization of the HPLC conditions where separation is obtained
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on Ci8 5 urn ODS Hypersil Column, flow rate is 1.0 ml / min and all separations are performed
at room temperature with detection at 210 nm wavelength. The short chromatographic time of
approximately 10 min makes this method suitable for the processing of multiple samples in a
limited amount of time.
INDUSTRIAL APPLICATION: The present method is simple, fast and cheap. The industrial
applications of the present invention lie in the robustness and ruggedness of the disclosed method
as compared with other existing HPLC methods. 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 of the drug.
PROCESS OF DETERMINATION:
The method involved in this process includes HPLC system and UV detector. The brief
description of the same is described below:
HPLC system: It is a form of column chromatography used frequently in biochemistry and
analytical chemistry to separate, identify and quantify compounds. HPLC utilizes a column that
holds chromatographic packing material (stationary phase), a pump that moves the mobile
phase(s) through the column and a detector that shows the retention times (The time interval
between the instant of injection and the detection of the component) of the molecules. Retention
time varies depending on the interactions between the stationary phase, the molecules being
analyzed and the solvent(s) used.
UV detector: UV detectors function on the capacity of many compounds to absorb light in the
wavelength range 180 to 350 nm. By measuring the absorbsion of compound at a specific
wavelength the concentration of the solute in the solution can be determined.
Preparation of standard stock solution: Pregabalin stock solution is prepared by dissolving an
appropriate amount of the compounds containing Pregabalin, Gabanext 75, Di- potassium
hydrogen orthophosphate and orthophosphoric acid in mobile phase to give a final concentration
of 0.75mg/ml. Standard solutions of pregabalin (2.0, 3.5 and 5.0 ug/ml) is prepared by
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subsequent dilution. A phosphate bufifer containing 0.02 M K2HP04 is added (3.50 gm in
1000 ml distilled water) and pH is adjusted to 7.0 by adding 0.01 M orthophosphoric acid.
Procedure for determination of pregabalin:
• Aliquots of stock solution (0.75 mg/ml) was transferred in to a 50 ml volumetric flask
and volumes are made up to the mark with the mobile phase (mixture of methanol -
acetonitrile - 0.02 M di-potassium hydrogen orthophosphate (pH -7.00) (3 : 1 : 16,
v/v/v) to produce solutions in the concentration range of 0.75 - 6.0 |^g/ml.
• 20 ul of the solution was injected into the HPLC system. The eluent was detected by the
UV detector at a wavelength of 210 nm.
• The signal emerging from the detector was integrated as peak area and a calibration
graph of peak area against the concentration of pregabalin was plotted. Alternatively,
the regression equation was derived.
Procedure for pharmaceutical formulations:
• 1 capsule of 75 mg of pregabalin was accurately weighed and finely powdered.
• 75 mg of PGB powder was extracted by shaking with 20 ml of the mobile phase,
followed by another 2 extractions each with 10 ml mobile.
• Solution was passed through a 0.45 urn Millipore filter and diluted with mobile phase to
obtain a concentration of about 0.75 mg/ml.
• Solution was further diluted according to need and then analyzed following proposed
procedures.
• The nominal content of PGB was determined from the corresponding regression
equation.
Procedure for the determination of pregabalin in human urine samples:
• Aliquot volume of human urine samples was transferred into small separating
funnel.
• 5 ml of carbonate buffer pH - 9.4 (prepared by dissolving 26.5 g sodium carbonate and
21.0 g sodium bicarbonate in 500 ml distilled water) was added and solution was
mixed properly.
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• The solution was extracted with 3 x 5 ml of diethyl ether and ether extract was collected
and evaporated.
• The residue was dissolved in 5 ml of mobile phase and above general procedure
was followed.
• The nominal content of PGB was determined from the corresponding regression
equation.
METHOD VALIDATION:
System stability: The system suitability of the proposed method was evaluated after spiking
PGB and closely eluted compounds such as 3-isobutyl glutaric acid and (R) - (-) - 3 -
(carbamoylmethyl) -5- methyl hexanoic acid. The resolution of PGB with 3 -isobutyl glutaric
acid and (R) - (-) - 3 - (carbamoylmethyl) - 5 - methyl hexanoic acid was found to be more than
2.
Determination of suitable UV wavelength: In order to investigate the appropriate
wavelength for the determination of PGB, solution of PGB in mobile phase was scanned
by UV spectroscopy in the range of 200 - 400 nm. The maximum absorbance was observed
at 210 nm. Alternatively, solution of PGB in the same mobile phase was also injected to HPLC
directly at different wavelength. But the maximum peak area was observed at 210 nm. Therefore,
it was conclude that 210 nm is the most appropriate wavelength for the analysis of PGB with
suitable sensitivity.
Specificity: The specificity and selectivity of the proposed method was evaluated by
estimating the amount of pregabalin in the presence of common excipients such as sodium
stearyl fumarate, magnesium stearate, starch, lactose glucose, talc and methyl cobalamin.
The HPLC chromatograms recorded for the mixture of the drug excipients revealed almost
no peaks within a retention time range of 10 min. The study of the absence of excipients showed
that none of the peaks appears at the retention time of PGB and it was concluded that the
developed method is selective in relation to the excipients of the final preparation.
Solution stability: The solution stability was ascertained from HPLC peak area of reference
standard samples. The peak area was obtained at 4.632 min retention time with a UV detector
of wavelength of 210 nm (2.000 AUFS). The standard sample solutions were kept at room
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temperature for 15 days, it was observed that there was no change in peak area of these
solutions.
Accuracy and precision: Under the optimum experimental conditions, the peak area -
concentration plot for the proposed method was found to be rectilinear over the range of
0.75 - 6.0 u.g/ml. Linear regression analysis of calibration data gave the regression equation
cited in Table 1 with correlation coefficient close to unity. Statistical analysis of regression line
was made regarding the standardbdeviation of residuals (So), standard deviation of slope
(Sb) and standard deviation of intercept (Sa) and the values are summarized in Table 1.
Table 1. Summary of optical and regression characteristics of the proposed method.
'With respect to Y = a + b X , where X is the concentration in ng/ml, Y is peak area; "Confidence interval of the intercept and slope at 95 %
confidence level and ten degrees of freedom (t = 2.228)
The days of precision assays were carried out through replicate analysis (n = 5) of PGB
corresponding to 2.0, 3.5 and 5.0 u.g/ml for the proposed method in pure form and
pharmaceutical formulations as shown in Table 2 and 3 below. The recovery and relative
standard deviation (RSD) by intraday and interday precision were in the ranges 99.990 -
100.056%, 0.036 - 0.167%, 99.98 - 99.99% and 0.068- 0.162% respectively as shown in Table2.
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Table 3 shows the recovery and RSD by intraday and interday precision which were in the
ranges 99.940 - 99.983 %; 0.174 - 0.785% and 99.927 - 99.970 %; 0.203 - 0.851%
respectively. The precision results were found to be satisfactory.
Table 2. Summary of accuracy and precision results of the proposed method in pure form
a Mean for 5 independent analyses.b SAE, standard analytical error. CC.L., confidence limit at 95 % confidence level and 4
degrees of freedom (t = 2.776). RSD; Related standard deviation.
Table 3. Summary of accuracy and precision results of the proposed method in
pharmaceutical formulations
" Mean for 5 independent analyses.b SAE, standard analytical error.c C.L., confidence limit at 95 % confidence level and 4
degrees of freedom (t = 2.776). RSD; Related standard deviation.
Table 4 given below shows the estimation of PGB from capsules after spiking with 1.0,2.5 and
4.0 ug/ml of additional pure drug and the recoveries obtained were ranged from 99.906 to
100.088% with relative standard deviation between 0.119 to 0.465%.
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Table 4. Summary of data for the determination of pregabalin in pharmaceutical preparations by
standard addition method
a Mean for 5 independent analyses.b SAE, standard analytical error.
Table 5 shows the in vitro determination of PGB in human urine samples and result was found
to be satisfactorily accurate and precise.
Table 5. Application of the proposed HPLC method to the determination of Pregabalin in human
urine samples
Robustness: The influences of small changes in the mobile phase composition and buffer pH
were studied to determine the robustness of the method, such as the changes in peak area and
retention time as shown in Table 6. The robustness of the method was also assessed by
analyzing the active PGB in pharmaceutical formulations. The reference standard sample
solution containing 5.0 ug/ml of the drug assayed and the percent recovery ± RSD of the method
(99.989 ± 0.150) were found to be appreciable, indicating that the proposed method is robust.
Table 6. The influence of Small Changes in pH and Composition of Mobile Phase (Method
Robustness)
Ruggedness: For the evaluation of ruggedness of the proposed method, the contents of PGB at
5.0 ug/ml were assayed following the recommended procedure using Auto sampler and Manual
HPLC systems. The recoveries ± RSD resulting from Auto sampler (99.985 ± 0.145) and Manual
HPLC systems (99.981 ± 0.252) were compared.
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In the preceding detailed description, the invention is described with reference to specific
exemplary embodiments thereof. Various modifications and changes may be made thereto
without departing from the broader spirit and scope of the invention as set forth in the claims.
The specification and data are, accordingly, to be regarded in an illustrative rather than a
restrictive sense. Thus, without further analysis, the foregoing will so fully reveal the gist of the
present invention that others can, by applying current knowledge, readily adapt it for various
applications without omitting features that, from the standpoint of prior art, fairly constitute
essential characteristics of the generic or specific aspects of this invention.
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Conclusion: From the above obtained data it can be concluded that the proposed HPLC method
a simple, accurate and reproducible method for routine in vitro tests of PGB in bulk
pharmaceutical formulations and human urine samples. The major advantages of this method
include
• short retention time without derivatization with other reagent
• stability of the solution
• no need for prior separation or purification before analysis and
• the applicability of a common HPLC system (isocratic system, UV detector).
The short chromatographic time makes this method suitable for the processing of multiple
samples in a limited amount of time. In addition, the method has wider linear dynamic range
with good accuracy and precision. The method shows no interference from common
excipients present in formulations. The statistical parameter and recovery data reveal the
good accuracy and precision of the proposed method. Finally, since no pharmacopoeial
method for determination of pregabalin in bulk and pharmaceutical formulations have been
reported yet, the proposed method could be very useful and suitable for the determination of
PGB in bulk, pharmaceuticals, formulations and human urine samples.

I CLAIM:
1) A novel process for pregabalin determination in bulk pharmaceuticals,
formulations and human urine samples with the help of High Performance Liquid
Chromatography system wherein:
- the determination can be carried out directly without the need for derivatization
- the detector system is ultra-violet detector
- the mobile phase used is a mixture of methanol - acetonitrile - di - Potassium
Hydrogen Orthophosphate in the ratio of 3 : 1 : 16, v/v/v
- the column used is a reverse phase column such as CI8
2) The novel process for pregabalin determination as claimed in claim 1 wherein
molarity of the mobile phase is optimally 0.02M.
3) The novel process for pregabalin determination as claimed in claim 1 wherein pH of
the mobile phase is optimally maintained at 7.00.
4) The novel process for pregabalin determination as claimed in claim 1 wherein
separation of solutions is obtained on a reverse phase column such as C18 5 urn
ODS Hypersil Column.
5) The novel process for pregabalin determination as claimed in claim 1 wherein flow
rate of the mobile phase solution is approximately 1.0 ml / min.
6) The novel process for pregabalin determination as claimed in claim 1 wherein the
quantiation of pregabalin using the UV detector is carried out at 210nm wavelength.
7) A process for the direct estimation of pregabalin in bulk pharmaceuticals,
formulations and human urine samples with the help of High Performance Liquid
Chromatography system using UV detector and without the need for derivatization
as described substantially herein with reference to the detailed description.