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Simultaneous determination of pseudoephedrine hydrochloride and diphenhydramine hydrochloride in cough syrup by gas chromatography (GC)
Talanta 46 (1998) 221 – 225
Simultaneous determination of pseudoephedrine hydrochloride and diphenhydramine hydrochloride in cough syrup by gas chromatography (GC) S.V. Raj, S.U. Kapadia, A.P. Argekar * Analytical Laboratory, Department Of Chemistry, The Institute of Science, 15, Madam Cama Road, Mumbai, 400 032, India Received 30 April 1997; received in revised form 12 August 1997; accepted 18 August 1997
Abstract A simple, rapid and precise gas chromatographic method has been developed for the simultaneous determination of pseudoephedrine hydrochloride and diphenhydramine hydrochloride in cough syrup, using a SS column of 10% OV 1 on chromosorb W-HP (80–100 mesh) and nitrogen as a carrier gas at a flow rate of 30 ml min − 1. The oven temperature was programmed at 135°C for 1 min, with a rise of 10°C min − 1 up to 250°C (held for 5 min). The injector and detector port temperatures were maintained at 280°C. Detection was carried out using Flame ionization detector. Guaphenesin was used as an internal standard. Results of assay and recovery studies were statistically evaluated for its accuracy and precision. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Simultaneous determination; Pseudoephedrine hydrochloride; Diphenhydramine hydrochloride; Cough syrup; Gas chromatography
1. Introduction Pseudoephedrine Hydrochloride (PSE), is a sympathomimetic agent. It is used for the symptomatic relief of nasal congestion. Diphenhydramine Hydrochloride (DPH), is an antihistamine with antimuscarinic and pronounced sedative properties. It is also used as an antiemetic [1]. Literature survey reveals that both PSE and
* Corresponding author.
DPH are official in U.S.P. [2], B.P. [3] and I.P. [4] Various Spectrophotometric [5,6] and HPLC [7] methods are reported for the individual determination of PSE and Spectrophotometric [8] and GC [9] methods are reported for the individual determination of DPH from its formulations. HPLC determination of DPH and Ephedrine Hydrochloride in biachuanpeng tablets [10] and HPLC determination of DPH and Ondansetron mixture in 0.9% NaCl injection [11] are also reported. A HPTLC [12] method has been reported for the simultaneous determination of PSE and DPH in combined dosage form. However instru-
0039-9140/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 3 9 - 9 1 4 0 ( 9 7 ) 0 0 2 7 7 - 4
S.V. Raj et al. / Talanta 46 (1998) 221–225
222 Table 1 Results of system suitability Sr. No.
Typical analytical parameters used in assay validation as per U.S.P. PSE 23
1
Accuracy (%)
2
Precision [R.S.D. (%)]
1.85
1.73 2.00
3 4 5
Limit of detection (mg) Limit of quantification (mg) Linearity (r)
0.5 2.0 0.99
0.4 — 1.5 — 0.99 —
6
Range (mg ml−1)
98.32
9
7 8
Theoretical plates Resolution factors
9
Tailing factor
mental facility of HPTLC being rare as compared to GC and HPLC and there is no method for the simultaneous determination of PSE and DPH by GC. So a new method for the simultaneous determination of PSE and DPH from cough syrup by GC technique is developed.
2. Experimental
2.1. Instrument CHEMITO 5210 gas chromatograph equipped with an Oracle 2 integrator and a flame ionization detector was used.
2.2. Carrier gas Nitrogen was used as a carrier gas at a flow rate of 30 ml min − 1.
2.3. Stationary phase An S.S. column (2.50 m in length and 2 mm id) of 10% OV 1 on Chromosorb W-HP (80–100 Mesh), manufactured by Sky Lab, Thane, Mumbai, was used as a stationary phase.
DPH
As per U.S.P. 23 requirements
98.19 90 – 110% of labeled amount
8 1
1
—
– –5 10 3500 4400 Normally not less than 500 2.1 2.0 Varies from Monograph to Monograph 2.4 2.1 Same as above
2.4. Sol6ents and chemicals Chromatography grade Chloroform and A.R. Grade Sodium Hydroxide were used.
2.5. Standard stock solution A combined standard solution of PSE (5 mg ml − 1) and DPH (5 mg ml − 1) were prepared in water. These Standards were procured from Merind, Mumbai, India and their purities were found to be 98.95 and 99.28% respectively.
2.6. Internal standard Guaphenesin standard of 98.89% purity, was procured from TATA Pharma, Patalganga, India. A solution of 5 mg ml − 1 was prepared in water.
2.7. Procedure for calibration In to a series of separating funnels aliquots of standard drug solution equivalent to 1–10 mg ml − 1 for PSE and 1–5.0 mg ml − 1 for DPH were taken. 1 ml of internal standard and 20 ml of 1 N NaOH were added and extracted with three quantities, each of 20 ml of chloroform. The chloroform layer was collected in a conical flask and
S.V. Raj et al. / Talanta 46 (1998) 221–225
223
Amount of drug (mg per 5 ml) =
RA/RI × C× D RB/RI × W
where RA = area of sample, RB =area of standard, RI = area of internal standard, C= Concentration of standard in mg ml − 1, D= dilution factor, W= weight of sample in mg.
2.9. Procedure for reco6ery studies
Fig. 1. Typical chromatogram of PSE and DPH.
evaporated till 5 ml solution was left. Further evaporation was done using nitrogen up to dryness. The residue was reconstituted in 2 ml of chloroform. All the operating chromatographic conditions were set and the instrument was stabilized for steady baseline. 2 ml of the solution were injected into the chromatographic system. The chromatograms were recorded and peak area ratios were computed.
To study the accuracy, reproducibility and precision of the above method, recovery of the added standard was studied at three different levels. Each level was repeated three times. A plot of the amount of the drug found by the proposed method (Y-axis) against the amount of the standard added (X-axis) was drawn. The intercept on the Y-axis indicates the amount of drug present per five ml of cough syrup. From the amount of drug found. Percentage recovery was calculated using the formula: N(%XY)− (%Y)(%X) %Recovery=
× 100 2
N(%X )− (%X)
2.8. Procedure for assay Ten grams of cough syrup was accurately weighed and then transferred into a separating funnel. To this 1 ml of internal standard solution and 20 ml of 1 N NaOH solution were added and proceed as described in the procedure for calibration. The amount of drug present in mg per 5 ml was calculated using the formula: Table 2 Results of GC assays from tablets Sr. No.
PSE (Label claim: 30 mg per 5 ml)
DPH (label claim: 15 mg per 5 ml)
1 2 3 4 5 Mean R.S.D. (%)
28.92 30.19 28.48 29.27 28.54 29.34 1.85
14.30 15.24 13.63 13.85 14.66 14.76 1.73
2
where X =amount of standard drug added, Y= amount of drug found by the proposed method, N=number of observations.
3. Results and discussion
3.1. System suitability To ascertain the resolution and reproducibility of the chromatographic method, system suitability tests were carried out on freshly prepared standard stock solution of PSE and DPH. The Parameters obtained are as shown in Table 1. These results are in concurrence with U.S.P. 23 requirements.
3.2. Chromatography 10% OV1 column gave a good separation of the drugs and the internal standard. Sharp and sym-
S.V. Raj et al. / Talanta 46 (1998) 221–225
224 Table 3 Results of recovery analysis Level No.
Amount of drug in mg per 5 ml of syrup
Pseudoephedrine hydrochloride 1 30 2 30 3 30 4 30 % recovery= 98.32% Diphenhydramine 1 2 3 4 % recovery= 98.19% a
hydrochloride 15 15 15 15
Amount of standard Amount of drug recovered in mg per 5 ml of syrupa added in mg
R.S.D. (%)
0 5 10 15
29.18 34.32 39.06 43.98
1.80 1.92 1.88 1.98
0 3 6 9
14.70 17.84 20.57 23.46
1.69 1.76 1.89 1.85
Average of three experiments.
metrical peaks were achieved by programming the oven temperature at 135°C for 1 min, with a rise of 10°C min − 1 up to 250°C (held for 5 min). The retention times of PSE and DPH were about 4.69 and 10.50 min respectively. Retention time of the internal standard was 8.00 min (Fig. 1).
3.3. Linearity, limit of detection and limit of quantification A linear relationship was obtained in the concentration range of 1 – 10 mg ml − 1 for PSE and 1–5 mg ml − 1 for DPH respectively. The calibration curves could be represented by the linear regression equations: YPSE = 8.1980X +3.9850
(r=0.999)
YDPH = 5.4006X +5.2358
(r = 0.998)
where Y= area and X =concentration of the drug in mg. The Limit of Detection (LOD) and the Limit of Quantification (LOQ) of PSE and DPH were calculated on the peak area using the following equations:
LOD= 3× N/B and LOQ= 10× N/B where N, the noise estimate, is the standard deviation of the peak areas (n= 5) of the respective drugs and B is the slope of the corresponding calibration curve. LOD and LOQ were 0.5 and 2.0 mg ml − 1 for PSE and 0.4 and 1.5 mg ml − 1 for DPH respectively.
3.4. Assay In a replicate analysis (n= 5) for the cough syrup (LUPIHIST, Batch No. 6001, date of manufacturing: Feb. 1996, manufactured by Lupin, Chikalthana, Aurangabad, India.), the amounts of PSE and DPH per 5 ml of the cough syrup found by the proposed method were 29.34 mg (R.S.D. = 1.85%) and 14.76 mg (R.S.D.=1.73%) respectively, as shown in Table 2. The low values of R.S.D. indicate that the method is precise and accurate. A blank run of the excepients present per 5 ml of cough syrup, such as Sodium Citrate (150 mg), ammonium chloride (150 mg) and menthol (0.01 ml) did not show any interference.
S.V. Raj et al. / Talanta 46 (1998) 221–225
225
3.5. Accuracy and precision
References
The percentage recoveries obtained for PSE and DPH were 98.32 and 98.19% respectively, which indicates that the method is precise, accurate and there is no interference due to excepients present in the cough syrup (Table 3).
[1] Martindale The Extra Pharmacopoeia, 31st ed. 1588, The Royal Pharmaceutical Society of Great Britain, 1996, p. 442. [2] The United States Pharmacopoeia, 534, 23rd ed. The United States Pharmacopoeial Convention, Inc., 1995, p. 532. [3] British Pharmacopoeia, 231, 563. HMSO, 1993, p. 147. [4] Indian Pharmacopoeia, 641 – 642. The Controller of Publications, Delhi, 1996, p. 259. [5] J.L. Murtha, T.N. Julian, G.W. Radebaugh, J. Pharm. Sci. 77 (8) (1988) 715 – 718. [6] H.S.I. Jan, M. Kolmanpunporn, Anal. Chim. Acta. 226 (11) (1989) 159 – 164t. [7] Abdel-Moety, O.A. Al-Deeb, N.A. Khattab, J. Liq. Chromatogr. 18 (20) (1995) 4127 – 4134. [8] B. Xu, T. Bi, Yaoxue Xuebao 24 (5) (1989) 360 – 365. [9] Y. Zhang, C. Yang, Zhongguo Yaoxue Zazhi (Ch.) 26 (3) (1991) 163 – 164. [10] Y. Yu, Yaowu Fenxi Zazhi 10 (6) (1990) 355 – 356. [11] L. Ye, J.T. Stewart, J. Liq. Chromatogr. Relat. Tech. 19 (5) (1996) 711 – 718. [12] J.E. Haky, D.A. Sherwood, S.T. Brenkan, J. Liq. Chromatogr. 12 (6) (1989) 907 – 917.
4. Conclusion The proposed GC method is accurate, precise and rapid for the simultaneous determination of PSE and DPH from cough syrup. Hence it can be easily and conveniently adopted for the routine quality control analysis.
Acknowledgements Authors are thankful to M/S Merind Limited, Mumbai, India, for providing the instrumental facilities.
.
.
Simultaneous determination of pseudoephedrine hydrochloride and diphenhydramine hydrochloride in cough syrup by gas chromatography (GC) S.V. Raj, S.U. Kapadia, A.P. Argekar * Analytical Laboratory, Department Of Chemistry, The Institute of Science, 15, Madam Cama Road, Mumbai, 400 032, India Received 30 April 1997; received in revised form 12 August 1997; accepted 18 August 1997
Abstract A simple, rapid and precise gas chromatographic method has been developed for the simultaneous determination of pseudoephedrine hydrochloride and diphenhydramine hydrochloride in cough syrup, using a SS column of 10% OV 1 on chromosorb W-HP (80–100 mesh) and nitrogen as a carrier gas at a flow rate of 30 ml min − 1. The oven temperature was programmed at 135°C for 1 min, with a rise of 10°C min − 1 up to 250°C (held for 5 min). The injector and detector port temperatures were maintained at 280°C. Detection was carried out using Flame ionization detector. Guaphenesin was used as an internal standard. Results of assay and recovery studies were statistically evaluated for its accuracy and precision. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Simultaneous determination; Pseudoephedrine hydrochloride; Diphenhydramine hydrochloride; Cough syrup; Gas chromatography
1. Introduction Pseudoephedrine Hydrochloride (PSE), is a sympathomimetic agent. It is used for the symptomatic relief of nasal congestion. Diphenhydramine Hydrochloride (DPH), is an antihistamine with antimuscarinic and pronounced sedative properties. It is also used as an antiemetic [1]. Literature survey reveals that both PSE and
* Corresponding author.
DPH are official in U.S.P. [2], B.P. [3] and I.P. [4] Various Spectrophotometric [5,6] and HPLC [7] methods are reported for the individual determination of PSE and Spectrophotometric [8] and GC [9] methods are reported for the individual determination of DPH from its formulations. HPLC determination of DPH and Ephedrine Hydrochloride in biachuanpeng tablets [10] and HPLC determination of DPH and Ondansetron mixture in 0.9% NaCl injection [11] are also reported. A HPTLC [12] method has been reported for the simultaneous determination of PSE and DPH in combined dosage form. However instru-
0039-9140/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 3 9 - 9 1 4 0 ( 9 7 ) 0 0 2 7 7 - 4
S.V. Raj et al. / Talanta 46 (1998) 221–225
222 Table 1 Results of system suitability Sr. No.
Typical analytical parameters used in assay validation as per U.S.P. PSE 23
1
Accuracy (%)
2
Precision [R.S.D. (%)]
1.85
1.73 2.00
3 4 5
Limit of detection (mg) Limit of quantification (mg) Linearity (r)
0.5 2.0 0.99
0.4 — 1.5 — 0.99 —
6
Range (mg ml−1)
98.32
9
7 8
Theoretical plates Resolution factors
9
Tailing factor
mental facility of HPTLC being rare as compared to GC and HPLC and there is no method for the simultaneous determination of PSE and DPH by GC. So a new method for the simultaneous determination of PSE and DPH from cough syrup by GC technique is developed.
2. Experimental
2.1. Instrument CHEMITO 5210 gas chromatograph equipped with an Oracle 2 integrator and a flame ionization detector was used.
2.2. Carrier gas Nitrogen was used as a carrier gas at a flow rate of 30 ml min − 1.
2.3. Stationary phase An S.S. column (2.50 m in length and 2 mm id) of 10% OV 1 on Chromosorb W-HP (80–100 Mesh), manufactured by Sky Lab, Thane, Mumbai, was used as a stationary phase.
DPH
As per U.S.P. 23 requirements
98.19 90 – 110% of labeled amount
8 1
1
—
– –5 10 3500 4400 Normally not less than 500 2.1 2.0 Varies from Monograph to Monograph 2.4 2.1 Same as above
2.4. Sol6ents and chemicals Chromatography grade Chloroform and A.R. Grade Sodium Hydroxide were used.
2.5. Standard stock solution A combined standard solution of PSE (5 mg ml − 1) and DPH (5 mg ml − 1) were prepared in water. These Standards were procured from Merind, Mumbai, India and their purities were found to be 98.95 and 99.28% respectively.
2.6. Internal standard Guaphenesin standard of 98.89% purity, was procured from TATA Pharma, Patalganga, India. A solution of 5 mg ml − 1 was prepared in water.
2.7. Procedure for calibration In to a series of separating funnels aliquots of standard drug solution equivalent to 1–10 mg ml − 1 for PSE and 1–5.0 mg ml − 1 for DPH were taken. 1 ml of internal standard and 20 ml of 1 N NaOH were added and extracted with three quantities, each of 20 ml of chloroform. The chloroform layer was collected in a conical flask and
S.V. Raj et al. / Talanta 46 (1998) 221–225
223
Amount of drug (mg per 5 ml) =
RA/RI × C× D RB/RI × W
where RA = area of sample, RB =area of standard, RI = area of internal standard, C= Concentration of standard in mg ml − 1, D= dilution factor, W= weight of sample in mg.
2.9. Procedure for reco6ery studies
Fig. 1. Typical chromatogram of PSE and DPH.
evaporated till 5 ml solution was left. Further evaporation was done using nitrogen up to dryness. The residue was reconstituted in 2 ml of chloroform. All the operating chromatographic conditions were set and the instrument was stabilized for steady baseline. 2 ml of the solution were injected into the chromatographic system. The chromatograms were recorded and peak area ratios were computed.
To study the accuracy, reproducibility and precision of the above method, recovery of the added standard was studied at three different levels. Each level was repeated three times. A plot of the amount of the drug found by the proposed method (Y-axis) against the amount of the standard added (X-axis) was drawn. The intercept on the Y-axis indicates the amount of drug present per five ml of cough syrup. From the amount of drug found. Percentage recovery was calculated using the formula: N(%XY)− (%Y)(%X) %Recovery=
× 100 2
N(%X )− (%X)
2.8. Procedure for assay Ten grams of cough syrup was accurately weighed and then transferred into a separating funnel. To this 1 ml of internal standard solution and 20 ml of 1 N NaOH solution were added and proceed as described in the procedure for calibration. The amount of drug present in mg per 5 ml was calculated using the formula: Table 2 Results of GC assays from tablets Sr. No.
PSE (Label claim: 30 mg per 5 ml)
DPH (label claim: 15 mg per 5 ml)
1 2 3 4 5 Mean R.S.D. (%)
28.92 30.19 28.48 29.27 28.54 29.34 1.85
14.30 15.24 13.63 13.85 14.66 14.76 1.73
2
where X =amount of standard drug added, Y= amount of drug found by the proposed method, N=number of observations.
3. Results and discussion
3.1. System suitability To ascertain the resolution and reproducibility of the chromatographic method, system suitability tests were carried out on freshly prepared standard stock solution of PSE and DPH. The Parameters obtained are as shown in Table 1. These results are in concurrence with U.S.P. 23 requirements.
3.2. Chromatography 10% OV1 column gave a good separation of the drugs and the internal standard. Sharp and sym-
S.V. Raj et al. / Talanta 46 (1998) 221–225
224 Table 3 Results of recovery analysis Level No.
Amount of drug in mg per 5 ml of syrup
Pseudoephedrine hydrochloride 1 30 2 30 3 30 4 30 % recovery= 98.32% Diphenhydramine 1 2 3 4 % recovery= 98.19% a
hydrochloride 15 15 15 15
Amount of standard Amount of drug recovered in mg per 5 ml of syrupa added in mg
R.S.D. (%)
0 5 10 15
29.18 34.32 39.06 43.98
1.80 1.92 1.88 1.98
0 3 6 9
14.70 17.84 20.57 23.46
1.69 1.76 1.89 1.85
Average of three experiments.
metrical peaks were achieved by programming the oven temperature at 135°C for 1 min, with a rise of 10°C min − 1 up to 250°C (held for 5 min). The retention times of PSE and DPH were about 4.69 and 10.50 min respectively. Retention time of the internal standard was 8.00 min (Fig. 1).
3.3. Linearity, limit of detection and limit of quantification A linear relationship was obtained in the concentration range of 1 – 10 mg ml − 1 for PSE and 1–5 mg ml − 1 for DPH respectively. The calibration curves could be represented by the linear regression equations: YPSE = 8.1980X +3.9850
(r=0.999)
YDPH = 5.4006X +5.2358
(r = 0.998)
where Y= area and X =concentration of the drug in mg. The Limit of Detection (LOD) and the Limit of Quantification (LOQ) of PSE and DPH were calculated on the peak area using the following equations:
LOD= 3× N/B and LOQ= 10× N/B where N, the noise estimate, is the standard deviation of the peak areas (n= 5) of the respective drugs and B is the slope of the corresponding calibration curve. LOD and LOQ were 0.5 and 2.0 mg ml − 1 for PSE and 0.4 and 1.5 mg ml − 1 for DPH respectively.
3.4. Assay In a replicate analysis (n= 5) for the cough syrup (LUPIHIST, Batch No. 6001, date of manufacturing: Feb. 1996, manufactured by Lupin, Chikalthana, Aurangabad, India.), the amounts of PSE and DPH per 5 ml of the cough syrup found by the proposed method were 29.34 mg (R.S.D. = 1.85%) and 14.76 mg (R.S.D.=1.73%) respectively, as shown in Table 2. The low values of R.S.D. indicate that the method is precise and accurate. A blank run of the excepients present per 5 ml of cough syrup, such as Sodium Citrate (150 mg), ammonium chloride (150 mg) and menthol (0.01 ml) did not show any interference.
S.V. Raj et al. / Talanta 46 (1998) 221–225
225
3.5. Accuracy and precision
References
The percentage recoveries obtained for PSE and DPH were 98.32 and 98.19% respectively, which indicates that the method is precise, accurate and there is no interference due to excepients present in the cough syrup (Table 3).
[1] Martindale The Extra Pharmacopoeia, 31st ed. 1588, The Royal Pharmaceutical Society of Great Britain, 1996, p. 442. [2] The United States Pharmacopoeia, 534, 23rd ed. The United States Pharmacopoeial Convention, Inc., 1995, p. 532. [3] British Pharmacopoeia, 231, 563. HMSO, 1993, p. 147. [4] Indian Pharmacopoeia, 641 – 642. The Controller of Publications, Delhi, 1996, p. 259. [5] J.L. Murtha, T.N. Julian, G.W. Radebaugh, J. Pharm. Sci. 77 (8) (1988) 715 – 718. [6] H.S.I. Jan, M. Kolmanpunporn, Anal. Chim. Acta. 226 (11) (1989) 159 – 164t. [7] Abdel-Moety, O.A. Al-Deeb, N.A. Khattab, J. Liq. Chromatogr. 18 (20) (1995) 4127 – 4134. [8] B. Xu, T. Bi, Yaoxue Xuebao 24 (5) (1989) 360 – 365. [9] Y. Zhang, C. Yang, Zhongguo Yaoxue Zazhi (Ch.) 26 (3) (1991) 163 – 164. [10] Y. Yu, Yaowu Fenxi Zazhi 10 (6) (1990) 355 – 356. [11] L. Ye, J.T. Stewart, J. Liq. Chromatogr. Relat. Tech. 19 (5) (1996) 711 – 718. [12] J.E. Haky, D.A. Sherwood, S.T. Brenkan, J. Liq. Chromatogr. 12 (6) (1989) 907 – 917.
4. Conclusion The proposed GC method is accurate, precise and rapid for the simultaneous determination of PSE and DPH from cough syrup. Hence it can be easily and conveniently adopted for the routine quality control analysis.
Acknowledgements Authors are thankful to M/S Merind Limited, Mumbai, India, for providing the instrumental facilities.
.
.