HPLC-UV Determination of Dextromethorphan in Syrup Method validation

NICOLAE AVRAM1, SIMONA CODRUTA HEGHES2, LUCA-LIVIU RUS3*, ANCA MARIA JUNCAN3, LUCIA MARIA RUS2, LORENA FILIP4, CORINA ROMAN FILIP5,6 1 S.C. Bioef S.R.L., Independent Drug Analysis Laboratory, 1 Ferma Str., Dostat, 517275, Alba, Romania 2 Iuliu Hatieganu University of Medicine and Pharmacy, Department of Drug Analysis, 6 Louis Pasteur Str., 400349, Cluj-Napoca, Romania 3 Lucian Blaga University, Faculty of Medicine, Preclinical Department, 2 A Lucian Blaga Str., 550169, Sibiu, Romania 4Iuliu Hatieganu University of Medicine and Pharmacy, Department of Bromatology, Hygiene, Nutrition, 8 Victor Babes Str., 400012, Cluj-Napoca, Romania 5Lucian Blaga University, Faculty of Medicine, Clinical Department, 2 A Lucian Blaga Str., 550169, Sibiu, Romania 6 Academic Emergency Hospital Sibiu, Department of Neurology, 2-4 Pompeiu Onofreiu Str., 550166, Sibiu, Romania

A HPLC-UV method, for determination of dextromethorphan hydrobromide in syrup, was validated. The chromatographic analysis was performed using an RP-18, Nucleodur chromatographic column (250 mm × 4 mm, 5 µm) at constant temperature (50 o C) with a mobile phase consisting of a mixture of acetonitrile/ methanol (70:30 v/v) with sodium docusate (as ion pair agent) and ammonium nitrate, pH = 3.4. The flow rate of the mobile phase was 1 mL/min and the detection was carried out at 280 nm. System suitability, specificity, linearity, precision, accuracy, limit of detection and limit of quantification agreed with current pharmacopeial requests. The method is suitable for routine analysis of dextromethorphan hydrobromide in syrup.
Keywords: dextromethorphan, HPLC, validation, syrup Dextromethorphan hydrodromide (DXHB) has been used as antitussive for more than half a century [1], single or in combinations [2]. DXHB is a non-selective serotonin uptake inhibitor and an agonist of sigma-1 receptor, and acts centrally to elevate threshold for coughing [3,4]. The activity of DXHB on N-methyl-D-aspartate receptor might have a contribution on antitussive effect, also for cought in palliative care [5,6].

Materials and methods
Standards, reagents and pharmaceutical substances Glycerol (batch 1555415) and pharmaceutical sugar (batch 7161T43034) were supplied by AAK Sweden AB, Sweden and Tereos, France, respectively. DXBH, batch 4, was supplied by EDQM.

Apparatus and chromatographic conditions
Ultrapure water was prepared by means of a Simplicity apparatus (Millipore), weighing was performed on a Mettler Toledo analytical balance, density (ρ s ) was determined by * email: liviu.rus@ulbsibiu.ro All the authors have equally contributed to this article. means of KEM digital densimeter, chromatographic analysis was performed on an Rigol L-3000, HPLC system (quaternary pump, autosampler, column oven, DAD detector, Clarity chromatographic software). Samples were injected into an RP-18, Nucleodur chromatographic column (250 mm × 4 mm, 5 µm) at constant temperature (50 o C). The mobile phase was prepared by solving 3.11 g of sodium docusate in a mixture consisting of 700 mL of acetonitrile and 300 mL of purified water, followed by the addition of 0.56 g of ammonium nitrate. The apparent pH was adjusted to 3.4 with glacial acetic acid. Injection volume was 20 µL, flowrate was 1 mL/min., detection wavelength was 280 nm.

Results and discussions
Synthetic mixtures of the drug product components, preparation and sample preparation In order to perform the validation of the method several synthetic mixtures of the drug product components (SMDPC), according to Annex 4 of the Marketing Authorization (MA) for the commercial product Tussin 6.5 mg/ 5 mL, syrup, considering different concentration levels of DXBH (0% -80%-90%-100%-110%-120%) as stated in table 1.
An appropriate amount (≈10 g) of SMDPC, all series, except serie 7, was weighed in 100 mL volumetric flasks. All flasks were filled to mark with purified water and sonicated for 5 minutes and a filtration through acrodisc filters was performed prior to injection. For all series, except serie 7, the relative density was determined.

Method validation
HPLC-UV method was validated in terms of system suitability, specificity, linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ).
The information presented in table 2 and figure 1, leds us to the conclusion that serie 1 showed no analytical signal, F calculated < F theoretical (the results are reproducible), t calculated < t theoretical (the means do not differ, statistically speaking), so developed method is specific.
The calibration curve parameters were calculated in Microsoft Excel, and the calibration curve is presented in equation (1) and r = 0.9997. A=7169.7 x C s -43.046 (1) The method proved to be linear in the range 0.0776-0.1163 mg/mL.
Precision of the method was proved by evaluating repeatability and reproducibility in three different days considering 100% concentration level (Series 8, 9 and 10), 5 replicates each day. Statistical analysis of precision consisted off: variances homogeneity evaluation (Cochrane test), repeatability variation coefficient (CV r %) and reproducibility variation coefficient (CV R %), considering a 5% error probability and 15 (5 × 3) samples.  As stated in table 4, all variances are homogeneous (since C calculated < C theoretical ), and both variation coefficients are below 2%. The method is precise.
Accuracy of the method was estimated by means of recovery using the same samples described in linearity testing (series 2, 3, 4, 5 and 6) in triplicate.  As shown in table 5, all variances are homogeneous (C calculated < C theoretical ), mean recoveries are valid (F calculated < F theoretical ) and confidence interval is very close to 100%. The method is accurate.  Table 6 LOD AND LOQ Limit of detection (LOD) and limit of quantification (LOQ) were determined based on signal-to-noise approach and are presented in table 6.

Conclusions
A HPLC-UV method, for determination of dextromethorphan hydrobromide in syrup, was validated in terms of system suitability, specificity, linearity, precision, accuracy, limit of detection and limit of quantification. All system suitability criteria were fullfilled. The method is: specific (results are reproducible and the means do not differ, statistically speaking), linear (over the concentration range 0.0776 -0.1163 mg/mL, correlation coefficient r = 0.9997), precise (all variances are homogeneous and both repeatability and reproducibility coefficients are below 2%), accurate (variances are homogeneous, mean recoveries are valid, and confidence interval is very close to 100%). LOD and LOQ are 4.142×10 -3 µg/mL and 1.38×10 -2 µg/ mL, respectively. The HPLC-UV is suitable for routine quantitative determination of dextromethorphan hydrobromide in syrup.