Gabapentin Assay: Ph. Eur. Monograph

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

Read more about

• Gabapentin Assay and System Suitability Test
• Assay of Gabapentin Related Substances A, B, and E and System Suitability Test
• Repeatability data and EP Criteria Gabapentin, Ph. Eur. Monograph
• Conclusion
• Related Products

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.

Figure 1. Blank chromatogram.

Figure 2. Reference solution C for System suitability.

Chromatographic Data - System Suitability Solution

Peaks	Compound	Retention Time (min)	Area	Tailing Factor	Tailing Factor (EP)
1	Gabapentin	4.615	4528082	3.07	max. 5.0

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

Chromatographic Data - Test Solution 14 mg/mL

Peaks	Compound	Retention Time (min)	Area	Tailing Factor	Tailing Factor (EP)
1	Gabapentin	4.621	4564753	3.2	max. 5.0

Gabapentin Assay - Calibration

Figure 4. Calibration function Gabapentin.

Table 1. Calibration data- Gabapentin

Conc. (µg/mL)	Area Response
0.35	284
0.7	343
1.4	893
3.5	2130
7.0	4020
10.5	6182
14.0	8115
17.5	10092
21.0	11902
Intercept	79.10
Slope	569.44
STEYX	95.48
LOD (µg/mL)	0.55
LOQ (µg/mL)	1.68

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.

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

Chromatographic Data – Reference solution A, 14 µg/mL

Peaks	Compound	Retention Time (min)	Tailing Factor	Area
1	Gabapentin	5.015	1.21	10072

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

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

Chromatographic Data – Reference Solution B

Peaks	Compound	Retention Time (min)	Tailing Factor	Area	Resolution	Resolution (EP)
1	Gabapentin Impurity A	14.305	1.09	62061	2.79	min. 2.3
2	Gabapentin Impurity B	15.476	1.04	18937

Table 2. Calibration Data- Gabapentin Related Substance A, B and E

Compound	Concentration range (µg/mL)	No. of calibrators	LOD (mg)mL)	LOQ (µg/mL)	R2
Impurity A	0.53-31.5	9	0.86	2.61	0.9996
Impurity B	1.4-21	7	2.0	6.06	0.9942
Impurity E	0.35 - 21	9	1.05	3.19	0.9985

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.

Figure 7. Reference Solution Gabapentin Related Substance D.

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)

Peaks	Compound	Retention Time (min)	Tailing Factor	Area
1	Gabapentin IMP D	11.358	1.06	79494

Table 3. Calibration Data- Gabapentin Related Substance D

Compound	Concentration range (µg/mL)	No. of calibrators	LOD (µg)mL)	LOQ (µg/mL)	R2
Impurity D	0.18 - 10.5	9	0.52	1.58	0.9985

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)	1.05	0.08
Reference Solution C (14 mg/ml Gabapentin)	0.09	0.04
Reference Solution D (7 µg/mL Impurity D)	0.53	0.04

System Suitability

Table 4. System Suitability - Data of Gabapentin Reference Solution A (14 mg/mL) using Purospher^® STAR RP-18e

Component	Ph Eur  Specification	Observed Value
% RSD Peak Area	NMT 2%	1.05%
% RSD Retention Time	NMT 1%	0.08%
Symmetry Factor	NMT 2	1.21
Resolution, Reference Solution B	Minimum 2.3	2.79

Table 5. System Suitability - Data of Gabapentin Reference Solution C (14 mg/mL) using Purospher^® STAR RP-18e

Component	Ph Eur  Specification	Observed Value
% RSD Peak Area	NMT 2%	0.09%
% RSD Retention Time	NMT 1%	0.04%
Symmetry Factor	NMT 5	3.2
% Assay Gabapentin Anhydrous Basis	97.5 to 102%	98.9%

Table 6. System Suitability - Data of Gabapentin Reference Solution D (7 µg/mL) using Purospher^® STAR RP-18e

Component	Ph Eur. Specification	Observed Value
% RSD Peak Area	NMT 2%	0.53%
% RSD Retention Time	NMT 1%	0.04%
Symmetry Factor	NMT 2	1.06

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

Table 7. Recovery of Gabapentin Related Impurity A

Gabapentin Related Imp A
No	Spike Level	µg/mL Added	µg/mL Found	% Recovery
1	LOQ	2.70	2.88	106.7
2	50%	10.50	11.54	109.9
3	100%	21.00	22.69	108.1
4	150%	31.50	32.61	103.5

Table 8. % Recovery of Gabapentin Related Impurity B

Gabapentin Related Imp B
No	Spike Level	µg/mL Added	µg/mL Found	% Recovery
1	LOQ	5.80	5.95	102.6
2	50%	7.00	7.18	102.6
3	100%	14.00	15.17	108.4
4	150%	21.00	21.57	102.7

Table 9. % Recovery of Gabapentin Related Impurity D

Gabapentin Related Imp D
No	Spike Level	µg/mL Added	µg/mL Found	% Recovery
1	LOQ	1.60	1.59	99.4
2	50%	3.50	3.53	100.9
3	100%	7.00	7.3	104.3
4	150%	10.50	10.5	100.0

Table 10. Recovery of Gabapentin Related Impurity E

Gabapentin Imp E
No	Spike Level	µg/mL Added	µg/mL Found	% Recovery
1	LOQ	3.2	3.51	109.7
2	50%	7.0	6.99	99.9
3	100%	14.0	14.01	100.1
4	150%	21.0	20.17	96.5

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

Table 11. Repeatability of Gabapentin Related Impurities at LOQ

Compound	Conc. (µg/mL)	%RSD (n=5)
Impurity A	2.6	1.28
Impurity B	6.0	1.06
Impurity D	1.6	1.50
Impurity E	3.2	0.49

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