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
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Experimental Conditions | |
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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.
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 |
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 |
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 |
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 |
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
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 |
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% |
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
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 |
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 |
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 |
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 | ||
---|---|---|
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
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