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HomeSmall Molecules Analysis & QCGabapentin Assay: Ph. Eur. Monograph

Gabapentin Assay: Ph. Eur. Monograph

Chemical structure of Gabapentin

Figure 1.Chemical structure of Gabapentin.

Gabapentin is an anticonvulsant medication primarily used to treat partial seizures and neuropathic pain. It is a first-line medication for the treatment of neuropathic pain caused by diabetic neuropathy, post-therapeutic neuralgia, and central neuropathic pain.1 It is moderately effective - about 30-40% of those given gabapentin for diabetic neuropathy or post-therapeutic neuralgia have a meaningful benefit. Gabapentin acts by decreasing the activity of a subset of calcium channels.2

We demonstrate the assay and related substances determination of gabapentin following the Ph. Eur monograph using the Purospher® STAR RP-18 end-capped column, 250 x 4.6 mm, 5 μm.3

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Gabapentin Assay and System Suitability Test

Experimental Conditions
Column:Purospher® STAR RP-18 endcapped, 250 x 4.6 mm, 5 µm (1.51456)
Buffer solution:Dissolve 0.58 g of ammonium dihydrogen phosphate and 1.83 g of sodium perchlorate in 950 mL of water. Adjust to pH 1.8 with perchloric acid and top to 1000 mL with water.
Mobile phase:Acetonitrile: buffer solution (24:76 V/V).
Flow rate:1 mL/min
Back pressure:180 bar (2600 psi)
Column temp.:40°C
Detection:UV, 215 nm
Injection volume:20 µL
Samples: 
Solution A (diluent):Dissolve 2.32 g of ammonium dihydrogen phosphate in 950 mL of water, adjust to pH 2.0 with phosphoric acid, and top to 1000 mL with water.
Test solution (14 mg/mL):Weigh and crush 10 tablets and transfer the powdered sample containing gabapentin equivalent to 0.140 g into a 10 mL volumetric flask. Add Solution A with intermittent swirling and sonication for about 10 mins and top up with Solution A. Filter through 0.45 µm PVDF syringe filter.
Reference solution C:Dissolve 0.140 g of gabapentin CRS in solution A and dilute to 10 mL with Solution A.
Blank chromatogram

Figure 1.Blank chromatogram.

Chromatogram of System suitability solution

Figure 2.Reference solution C for System suitability.

Chromatographic Data - System Suitability Solution

PeaksCompoundRetention Time (min)AreaTailing FactorTailing Factor (EP)
1Gabapentin4.61545280823.07max. 5.0
Chromatogram of Gabapentin test solution (14mg/mL)

Figure 3.Chromatogram of Gabapentin test solution (14mg/mL).

Chromatographic Data - Test Solution 14 mg/mL

PeaksCompoundRetention Time (min)AreaTailing FactorTailing Factor (EP)
1Gabapentin4.62145647533.2max. 5.0

Gabapentin Assay - Calibration

Calibration curve obtained for Gabapentin in its assay

Figure 4.Calibration function Gabapentin.

Conc. (µg/mL)Area Response
 0.35284
 0.7343
 1.4893
 3.52130
 7.04020
10.56182
14.08115
17.510092
21.011902
Intercept79.10
Slope569.44
STEYX95.48
LOD (µg/mL)0.55
LOQ (µg/mL)1.68
Table 1.Calibration data- Gabapentin

Assay of Gabapentin Related Substances A, B, and E and System Suitability Test

Experimental Conditions 
Column:Purospher® STAR RP-18 endcapped, 250 x 4.6 mm, 5 μm (1.51456)
Buffer solution:Dissolve 0.58 g of ammonium dihydrogen phosphate and 1.83 g of sodium perchlorate in 950 mL of water. Adjust to pH 1.8 with perchloric acid and top to 1000 mL with water.
Mobile phase:           Acetonitrile: buffer solution (24:76 V/V)
Flow rate:1 mL/min
Back pressure:180 bar (2600 psi)
Column temperature:40°C
Detection:UV @ 215 nm
Injection volume:20 µL
Samples 
Solution A (diluent):Dissolve 2.32 g of ammonium dihydrogen phosphate in 950 mL of water. Adjust to pH 2.0 with phosphoric acid and top to 1000 mL with water.
Test solution (14 mg/mL):Weigh and crush 10 tablets and transfer powdered sample containing gabapentin equivalent to 0.140 g into a 10 mL volumetric flask. Add Solution A with intermittent swirling and sonication for about 10 mins and top up with Solution A. Filter through 0.45 µm PVDF syringe filter.
Reference solution A:Dilute 1 mL of the Test Solution to 100 mL with Solution A. Dilute 1 mL of this solution to 10 mL with Solution A.
Reference solution B:Dissolve 7 mg of gabapentin impurity A CRS and 10 mg of gabapentin impurity B CRS in 50 mL of methanol. Dilute 1 mL of this solution to 10 mL with Solution A.
Reference solution C:Dissolve 0.140 g of gabapentin CRS in Solution A and dilute to 10 mL with Solution A.
Chromatogram of Reference Solution A (14 mg/mL)

Figure 5.Reference solution A (14 mg/mL Gabapentin).

Chromatographic Data – Reference solution A, 14 µg/mL

PeaksCompoundRetention Time (min)Tailing FactorArea
1Gabapentin5.0151.2110072

Run time for impurity testing to be 4x retention time of Gabapentin = 20.06 min.

Chromatogram of Reference Solution B (Gabapentin Impurity A and B)

Figure 6.Reference solution B - Gabapentin impurity A and B.

Chromatographic Data – Reference Solution B

PeaksCompoundRetention Time (min)Tailing FactorAreaResolutionResolution (EP)
1Gabapentin Impurity A14.3051.09620612.79min. 2.3
2Gabapentin Impurity B15.4761.0418937
CompoundConcentration range (µg/mL)No. of calibratorsLOD (mg)mL)LOQ (µg/mL)R2
Impurity A0.53-31.590.862.610.9996
Impurity B1.4-2172.06.060.9942
Impurity E0.35 - 2191.053.190.9985
Table 2.Calibration Data- Gabapentin Related Substance A, B and E

Assay of Gabapentin Related Substance D

Experimental Conditions 
Column:Purospher® STAR RP-18 endcapped, 250 x 4.6 mm, 5 µm (1.51456)
Buffer solution:Dissolve 0.58 g of ammonium dihydrogen phosphate and 1.83 g of sodium perchlorate in 950 mL of water, adjust to pH 1.8 with perchloric acid and top to 1000 mL with water
Mobile phase:Methanol : acetonitrile : buffer solution (30:35:35 V/V/V)
Flow rate:1 mL/min
Back pressure:180 bar (2600 psi)
Column temperature:40°C
Detection:UV @ 215 nm
Injection volume:20 µL
Samples 
Solution A (diluent):Dissolve 2.32 g of ammonium dihydrogen phosphate in 950 mL of water. Adjust to pH 2.0 with phosphoric acid and top to 1000 mL with water.
Test solution:Weigh and crush 10 tablets and transfer powdered sample containing gabapentin equivalent to 0.140 g to 10 mL volumetric flask. Add Solution A with intermittent swirling and sonication for about 10 mins and top up with Solution A. Filter through 0.45 µm PVDF syringe filter.
Reference solution (D):Dissolve 7 mg of gabapentin impurity D CRS in 25 mL methanol and dilute to 100 mL with solution A. Dilute 1 mL of this solution to 10 mL with Solution A.
Chromatogram of Reference Solution OF Gabapentin Related Substance D

Figure 7.Reference Solution Gabapentin Related Substance D.

Test Solution: 14 mg/mL with Impurity D: 7 µg/mL

Figure 8.Test Solution 14 mg/mL substance showing 7.0 µg/mL Impurity D.

Chromatographic Data (Test Solution: 14 mg/mL Substance Showing 7.0 µg/ mL Impurity D)

PeaksCompoundRetention Time (min)Tailing FactorArea
1Gabapentin IMP D11.3581.0679494
CompoundConcentration range (µg/mL)No. of calibratorsLOD (µg)mL)LOQ (µg/mL)R2
Impurity D0.18 - 10.590.521.580.9985
Table 3.Calibration Data- Gabapentin Related Substance D

Repeatability data and EP Criteria Gabapentin, Ph. Eur. Monograph

Solution%RSD Area (n=5)%RSD RT (n=5)
Reference Solution A (14 µg/ml Gabapentin)1.050.08
Reference Solution C (14 mg/ml Gabapentin)0.090.04
Reference Solution D (7 µg/mL Impurity D)0.530.04

System Suitability

Component Ph Eur  SpecificationObserved Value
% RSD Peak Area NMT 2%1.05%
% RSD Retention TimeNMT 1% 0.08%
Symmetry FactorNMT 21.21 
Resolution, Reference Solution BMinimum 2.32.79
Table 4.System Suitability - Data of Gabapentin Reference Solution A (14 mg/mL) using Purospher® STAR RP-18e
ComponentPh Eur  SpecificationObserved Value
% RSD Peak AreaNMT 2%  0.09%
% RSD Retention TimeNMT 1%  0.04%
Symmetry FactorNMT 5  3.2
% Assay Gabapentin Anhydrous Basis97.5 to 102%98.9%
Table 5.System Suitability - Data of Gabapentin Reference Solution C (14 mg/mL) using Purospher® STAR RP-18e
ComponentPh Eur. SpecificationObserved Value
% RSD Peak AreaNMT 2%0.53%
% RSD Retention TimeNMT 1%0.04%
Symmetry FactorNMT 21.06
Table 6.System Suitability - Data of Gabapentin Reference Solution D (7 µg/mL) using Purospher® STAR RP-18e

Recovery of Gabapentin Related Substances A, B, D and E Spiked in Test solution (14 mg/mL), using Purospher® STAR RP-18e

Gabapentin Related Imp A
NoSpike Levelµg/mL Addedµg/mL Found% Recovery
1LOQ  2.70  2.88106.7
250%10.5011.54109.9
3100%21.0022.69108.1
4150%31.5032.61103.5
Table 7.Recovery of Gabapentin Related Impurity A
Gabapentin Related Imp B
NoSpike Levelµg/mL Addedµg/mL Found% Recovery
1LOQ  5.80  5.95102.6
250%  7.00  7.18102.6
3100%14.0015.17108.4
4150%21.0021.57102.7
Table 8.% Recovery of Gabapentin Related Impurity B
Gabapentin Related Imp D
NoSpike Levelµg/mL Addedµg/mL Found% Recovery
1LOQ  1.60 1.59  99.4
250%  3.50 3.53100.9
3100%  7.00  7.3104.3
4150%10.5010.5100.0
Table 9.% Recovery of Gabapentin Related Impurity D
Gabapentin Imp E
NoSpike Levelµg/mL Addedµg/mL Found% Recovery
1LOQ  3.2  3.51109.7
250%  7.0  6.99  99.9
3100%14.014.01100.1
4150%21.020.17  96.5
Table 10.Recovery of Gabapentin Related Impurity E

Repeatability of Gabapentin Related Substances at LOQ levels using Purospher® STAR RP-18e

CompoundConc. (µg/mL)%RSD (n=5)
Impurity A 2.61.28
Impurity B6.01.06
Impurity D1.61.50 
Impurity E3.20.49
Table 11.Repeatability of Gabapentin Related Impurities at LOQ

Conclusion

The assay and related substances determination of gabapentin in pharmaceutical preparations with the Ph. Eur. monograph3 is demonstrated with the Purospher® Star RP-18 endcapped column, 250 X 4.6 mm, 5 μm.

The assay method shown, provided for gabapentin a Limit of Detection (LOD) of 0.55 µg/mL, and a Limit of Quantification (LOQ) of 1.68 µg/mL with linearity to a concentration of up to 21.0 µg/mL. The RSD on the repeatability of the gabapentin assay was <2%.

For the related substances A, B, D, and E of gabapentin, the Limit of Detection (LOD) ranged from 0.52 to 2.0 µg/mL and the Limit of Quantification (LOQ) was 1.58 to 6.06 µg/mL. Percentage recoveries ranged from 96.5 to 109.9%.

See more information on quality control testing at SigmaAldrich.com/PharmaQC

References

1.
Attal N, Cruccu G, Baron R, Haanpää M, Hansson P, Jensen TS, Nurmikko T. 2010. EFNS guidelines on the pharmacological treatment of neuropathic pain: 2010 revision. Euro J of Neurology. 17(9):1113. https://doi.org/10.1111/j.1468-1331.2010.02999.x
2.
Wiffen PJ, Derry S, Bell RF, Rice AS, Tölle TR, Phillips T, Moore RA. Gabapentin for chronic neuropathic pain in adults. 2020(2): https://doi.org/10.1002/14651858.cd007938.pub4
3.
Ph. Eur. 11, 2173E. [Internet]. Available from: https://www.edqm.eu/en/european-pharmacopoeia-ph.-eur.-11th-edition
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