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Dextromethorphan is an anti tussive (cough suppressant) drug.

It is one of the active ingredients in many over the-counter cold and cough medicines, such as Mucinex

DM, Robitussin, NyQuil, Dimetapp, Vicks, Coricidin, Delsym, and others, including generic labels. Dextromethorphan has also found other uses in medicine, ranging from pain relief to psychological applications. It is sold in syrup, tablet, spray, and lozenge forms. In its pure form, dextromethorphan occurs as a white powder.[2]

Figure 1: Stricture of Dextromethorphan DXM is also used recreationally. When exceeding label-specified maximum dosages, dextromethorphan acts as a dissociative hallucinogen. Its mechanism of action is via multiple effects, including actions as a nonselective serotonin reuptake inhibitor[3] and a sigma-1 receptor[4][5]agonist and the action of its major metabolite dextrorphan as an NMDA receptor antagonist, producing effects similar to well those as the of the active controlled metabolite 3substances ketamine and phencyclidine (PCP),[6] as dextrorphan but below dextromethorphan itself.[7] Side-effects of dextromethorphan use can include At normal doses dextromethorphan cause side effects like Sudden infant death syndrome, body rash/itching, nausea, drowsiness, dizziness, Closed-eye hallucination, Difficulty breathing ect. Quinidine is a pharmaceutical agent that acts as a class I antiarrhythmic agent (Ia) in the heart. It is a stereoisomer of quinine, originally derived from the bark of the cinchona tree. quinidine primarily works by blocking the fast inward sodium current (INa). Quinidine's effect on INa is known as a use dependent block. This means that at higher heart rates, the block increases, while

methoxymorphinan, which produces local anesthetic effects in rats with a potency above

at lower heart rates the block decreases. The effect of blocking the fast inward sodium current causes the phase 0 depolarization of the cardiac action potential to decrease (decreased Vmax).

Figure 2 : Stricture of Quinidine At micromolar concentrations, quinidine inhibits Na/K-ATPase by binding to the same receptor sites as the digitalis glycosides such as ouabain. The effect of quinidine on the ion channels is to prolong the cardiac action potential, thereby prolonging the QT interval on the surface ECG. Other ECG effects include a wide notched P wave, wide QRS complex, depressed ST segment, and U waves. These are the results of both slowed depolarization and repolarization.[8] Quinidine is also an inhibitor of the cytochrome P450 enzyme 2D6, and can lead to increased blood levels of lidocaine, Beta blockers, opioids, and some anti-depressants. Quinidine also inhibits the transport protein P-glycoprotein and so can cause some peripherally acting drugs such as loperamide to have CNS side effects such as respiratory depression if the two drugs are co-administered.[9] Quinidine can cause thrombocytopenia, granulomatous hepatitis, myasthenia gravis, and torsades de pointes and for that reason is not used much today. Torsades can occur after the first dose. Quinidine-induced thrombocytopenia (low platelet count) is mediated by the immune system, and may lead to thrombocytic purpura. Quinidine intoxication can lead to a collection of symptoms collectively known as cinchonism with tinnitus (ringing in the ears) being among the most characteristic and common symptoms of this toxicity syndrome.

Experimental

Chemicals and Reagents Dextromethorphan and Quinidine as pure standard reference drugs were purchased from Natco Laboratory, Hyderabad and pharmaceutical formulation from local market were used for this present study. Water, Acetonitrile, methanol and orthophosphoric acid,Tri Ethyl Amine (all HPLC grade) were purchased from Merck Specialties Private Limited, Mumbai, India.

Instrumentation To develop a High Pressure Liquid Chromatographic method for quantitative estimation of Quinidine and Dextromethorphan, an isocratic PEAK HPLC instrument with Hypersil C18 column (250 mm x 4.6 mm, 5) was used. The instrument is equipped with a LC 20AT pump for solvent delivery and variable wavelength programmable LC 7000 UV-detector. A 20L Rheodyne inject port was used for injecting the samples. Data was analyzed by using PEAK software. UV-2306 Spectrophotometer was used for wavelength checking. Denver analytical Balance was used to weigh the drug.

Experimental Condition Flow rate of the mobile phase was changed from 0.5 1.5 ml/min for optimum separation. A minimum flow rate as well as minimum run time gives the maximum saving on the usage of solvents. It was found from the experiments that 1.0 ml/min flow rate was ideal for the successful elution of the analyte. The HPLC system was hence operated using an isocratic mode at a flow rate of 1.0 ml/min at 25 2c. For analysis the most suitable mobile phase was found to be Methanol, Acetonitrile, water and Ortho phosphoric acid in the ratio of 35:25:25:15. Detection was carried out at wavelength of 259 nm.

Preparation of Mobile Phase For the preparation of mobile phase suitable for the present determination methanol, acetonitrile water, Ortho phosphoric acid of HPLC grade were mixed, filtered and degassed in such a way that the final volume consisted of these in the ratio 35:25:25:15 respectively, whose pH was adjusted to 5.7.

Preparation of mixed standard solution Dextromethorphan and Quinidine (1mg/ml) standard stock solutions were prepared using methanol as a solvent. Aliquots of mixed standard solutions of Dextromethorphan and Quinidine were diluted in mobile phase to get a final concentration of 50-100ppm.

Preparation of sample solution of pharmaceutical formulation Pharmaceutical form containing 20 mg of Dextromethorphan and 10 mg of Quinidine was weighed and dissolved in 25 ml of methanol and sonicated for 15 min. Using methanol the volume was made up to 50 ml and filtered through 0.45 membrane filter. The final mixed sample solution corresponding to 70 ppm of Dextromethorphan and 70 ppm of Quinidine was prepared.

Recording of chromatograms After stabilization of the base line with the optimized chromatographic conditions standard solutions containing 50-100 ppm of Dextromethorphan and Quinidine were injected and the corresponding chromatograms were recorded. Retention time of Dextromethorphan and Quinidine were found to be 3.8 and 5.8 mins respectively. Likewise for sample solution chromatograms were recorded. Calibration curves were plotted using peak area retentions of standard drug peaks against concentration of corresponding standard solutions.

RESULTS AND DISCUSSION

Method validation The method was validated by determining linearity, precision, accuracy, specificity, ruggedness and robustness by analyzing 40-100 ppm of both Dextromethorphan and Quinidine.

S.NO

TEST H.P.L.C CONDITIONS ELUTION A.P.I CONC MOBILE PHASE PH COLUMN WAVE LENGTH FLOW RUNTIME RETENSION TIME AREA TH.PLATES TAILING FACTOR PUMP PRESURE

RESULT

1 2 3 4 5 6 7 8 9 10 11 12 13

ISOCRATIC 70ppm MeOH:ACN:Water:0.1%OPA 35:25:25:15 5.7 C18 259nm 1ml\min 10min Dextromethorphan 3.85 Quinidine 5.86 Dextromethorphan 310795 Quinidine 393719 Dextromethorphan 6871 Quinidine 15892 Dextromethorphan 1.32 Quinidine 1.42 9.9Psi

Table 1: Optimized chromatographic conditions for estimation of Dextromethorphan and Quinidine

Linearity The linearity of the response for Dextromethorphan and Quinidine assay method was determined by preparing and injecting standard solutions of Dextromethorphan and Quinidine. The linear regression data for the calibration curves indicate that the response is linear over the concentration range studied with correlation coefficient (r2) value, slope and intercept as shown in table 3.

600000 500000 400000 300000 200000 100000 0 0 -100000 20 40 60 80 100 120

Graph 1: Calibration Plot for Dextromethorphan and Quinidine

S.NO

CONC IN PPM 50 60 70 80 90 100

Dextromethorphan Peak Area 223997 266812 310795 351122 401004 436135 Table.2

Quinidine Peak Area 274314 331529 393719 448313 493865 557465

1 2 3 4 5 6

Parameters Calibration range (ppm) Slope Intercept Correlation coefficient (r2)

Dextromethorphan 50-100 4388.38 2156.07 0.9997

Quinidine 50-100 ppm 5565.084 -661 0.9997

Table 3: Regression analysis of the calibration curve

Precision The precision of the assay was studied with respect to both repeatability and intermediate precision. Repeatability was calculated from six replicate injections of freshly prepared Dextromethorphan and Quinidine combined test solution in the same equipment at a concentration value of 70 ppm on the same day. The experiment was repeated by assaying freshly prepared solution at the same concentration additionally on two consecutive days to determine intermediate precision. Peak areas of the drugs were determined and precision as % RSD was reported. INJECTION 1 2 3 4 5 6 Dextromethorphan Quinidine 303543 299056 299514 300527 298843 299353 402542 398685 399732 398409 398746 399376 % of RSD Dextromethorphan % OF RSD Quinidine

0.59

0.4

Table.4 Intraday precision INJECTION 1 2 3 4 5 6 Dextromethorphan Quinidine 310795 300330 302517 300476 298864 301826 393719 396819 393719 398388 396689 398570 % of RSD Dextromethorphan % OF RSD Quinidine

0.14

0.45

Table.5 Interday precision

Parameters Theoretical plates (N) Retention time (min) Tailing factor LOD (ppm) LOQ (ppm)

Olmesartan 6871 3.8 1.32 0.5 1.6

Quinidine 15892 5.8 1.42 0.2 0.6

Table 6.System suitability and validation parameters

Figure.2 Typical chromatogram of standard Dextromethorphan and Quinidine

Recovery The recovery of the standard solutions was done by adding them to pre-analyzed sample solution at different levels i.e. 50%, 100%, and 150% separately to study the accuracy of the above method. The corresponding results were recorded.

S.NO

CONCENTRATI ON In ppm

Dextromethorp han AMOUNT RECOVERED

Quinidine AMOUNT RECOVERED

%of Dextromethorp han RECOVERED

% of Quinidine RECOVERED

1 2 3

50 75 100

49.87 75.12 100.055

50.02 74.96 100.255

99.74 100.16 100.055 Avg Recovery =99.985

100.04 99.95 100.025 Avg =100.005 Recovery

Table.7 Recovery of Dextromethorphan, Quinidine Specificity Specificity was performed to exclude the possibility of interference with excipients in the region of elution of Dextromethorphan and Quinidine. The specificity and selectivity of the method was tested under normal conditions and the results of the tests proved that the components other than the drug did not produce a detectable signal at the retention place of Dextromethorphan and Quinidine.

Limit of detection (LOD) and limit of quantification (LOQ) LOD and LOQ were determined from standard deviation of y-intercept of regression line and slope method as per ICH guidelines.

Robustness Typical variations in liquid chromatography conditions were used to evaluate the robustness of the assay method. In this study, the chromatographic parameters monitored were retention time, area, capacity factor, tailing factor and theoretical plates. The robustness acceptance criteria set in the validation were the same established on system suitability test describe above.

Parameter

Modification

Dextromethorpha n Area 310795 312161 309054 312369

Standard MP PH Wavelength

----------------------------------MeOH:ACN:Water:0.1%OP A 25:30:30:15 5.2 254nm

Quinidin e Area 393719 392930 394942 393085

Dextromethorph an % of change ....... 0.45 0.56 0.506

Quinidine % of change 0.25 0.31 0.16

Table.8 Robustness study

Analysis of marketed formulations The validated HPLC method was adopted for the quantification of Dextromethorphan and Quinidine in their combined pharmaceutical dosage form and the typical chromatograms of the formulation are shown in fig. The results of analysis are given in Table 8. The contents of the pharmaceutical dosage form were found to be in the range of 1002% with RSD less than 2% which indicate suitability for routine analysis of Dextromethorphan and Quinidine in pharmaceutical dosage form.

S.NO

Drug

Dosage

Sample Conc.

Amount drug estimated

of % of drug estimated

1 2

Dextromethorphan Quinidine

20 mg 10 mg

70 ppm 70 ppm

69.58 ppm 69.88 ppm

99.4 99.83

Table.9 Formulation

Conclusion The proposed study describes a new RP-HPLC method using simple mobile phase for the estimation of Dextromethorphan and Quinidine in combined pharmaceutical dosage formulations. The method was validated and found to be simple, sensitive, accurate and precise. It was also proved to be convenient and effective for the determination of Dextromethorphan and Quinidine in the pharmaceutical dosage form. The percentage of recovery shows that the method

is free from interference of the excipients used in formulation. Moreover, the lower solvent consumption along with the short analytical run time leads to cost effective chromatographic method. References 1. Kukanich, B.; Papich, M. G. (2004). "Plasma profile and pharmacokinetics of dextromethorphan after intravenous and oral administration in healthy dogs". Journal of Veterinary Pharmacology and Therapeutics 27 (5): 33741. 2. "Reference Tables: Description and Solubility - D". Retrieved 2011-05-06. 3. Schwartz AR, Pizon AF, Brooks DE (September 2008). "Dextromethorphan-induced serotonin syndrome". Clinical Toxicology (Philadelphia, Pa.) 46 (8): 7713. 4. Shin EJ, Nah SY, Chae JS, Bing G, Shin SW, Yen TP, Baek IH, Kim WK, Maurice T, Nabeshima T, Kim HC (May 2007). "Dextromethorphan attenuates trimethyltin-induced neurotoxicity via sigma1 receptor activation in rats". Neurochemistry International 50 (6): 7919. 5. Shin EJ, Nah SY, Kim WK, Ko KH, Jhoo WK, Lim YK, Cha JY, Chen CF, Kim HC (April 2005). "The dextromethorphan analog dimemorfan attenuates kainate-induced seizures via sigma1 receptor activation: comparison with the effects of dextromethorphan". British Journal of Pharmacology 144 (7): 90818. 6. "Dextromethorphan". Drugs Administration. August 2010. 7. Hou, C; Tzeng, J; Chen, Y; Lin, C; Lin, M; Tu, C; Wang, J (2006). "Dextromethorphan, 3-methoxymorphinan, and dextrorphan have local anaesthetic effect on sciatic nerve blockade in rats". European Journal of Pharmacology 544 (1-3): 106 8. Bowman, IA (1987). "Jean-Baptiste Senac and His Treatise on the Heart". Texas Heart Institute journal / from the Texas Heart Institute of St. Luke's Episcopal Hospital, Texas Children's Hospital 14 (1): 511. 9. Sadeque AJ, Wandel C, He H, Shah S, Wood AJ (2000). "Increased drug delivery to the brain by P-glycoprotein inhibition". Clin. Pharmacol. Ther. 68 (3): 2317 and Chemicals of Concern. Drug Enforcement