ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF LORNOXICAM AND THIOCOLCHICOSIDE IN BULK FORM AND MARKETED PHARMACEUTICAL DOSAGE FORM BY USING RP-HPLC

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS ESTIMATION OF LORNOXICAM AND THIOCOLCHICOSIDE IN BULK FORM AND MARKETED PHARMACEUTICAL DOSAGE FORM BY USING RP-HPLC

Dillip Kumar Jena

Gayatri Institute of Science and Technology, Gunupur, Rayagada - 765022, Odisha, India.

ABSTRACT: A simple, reproducible, and efficient reverse phase high performance liquid chromatographic method was developed for simultaneous determination of Lornoxicam and Thiocolchicoside in pure form and marketed combined pharmaceutical dosage forms. A column having Symmetry (C18) (150mm × 4.6mm, 5µm) in isocratic mode with mobile phase containing Methanol: Phosphate Buffer (pH-3.8) (28:72 v/v) was used. The flow rate was 1.0 ml/min, and the effluent was monitored at 252 nm. The retention time (min) and linearity range (ppm) for Lornoxicam and Thiocolchicoside were (1.794, 3.440 min) and (10-30, 10-50), respectively. The method has been validated for linearity, accuracy, and precision, robustness, and limit of detection, and limit of quantitation. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.86µg/ml and 2.58µg/ml for Lornoxicam and 1.28µg/ml 3.84µg/ml for Thiocolchicoside respectively. The developed method was found to be accurate, precise and selective for simultaneous determination of Lornoxicam and Thiocolchicoside in tablets.

Lornoxicam and Thiocolchicoside, RP-HPLC, Validation, Accuracy, Robustness

INTRODUCTION: Lornoxicam is an orally bioavailable oxicam and non-steroidal anti-inflammatory drug (NSAID), with analgesic, anti-pyretic, anti-thrombotic, and anti-inflammatory activities. Upon oral administration, Lornoxicam ²⁰ binds to and inhibits the activity of the cyclooxygenase enzymes (COX) type 1 (COX-1) and type 2 (COX-2). This blocks COX-mediated signaling pathways, which leads to reduced prostaglandin and thromboxane production and decreased pain, fever, and inflammation. Lornoxicam differs from other oxicam compounds in its potent inhibition of prostaglandin biosynthesis, a property that explains the particularly pronounced efficacy of the drug.

Lornoxicam is approved for use in Japan. The IUPAC Name ²¹ of Lornoxicam is 6-chloro-4-hydroxy-2-methyl-1, 1-dioxo-N-(pyridin-2-yl)-2H-1λ⁶-thieno [2, 3-e] [1,2] thiazine-3-carboxamide and the chemical formula ²² is C₁₃H₁₀ClN₃O₄S₂. The Chemical Structure of Lornoxicam is in Fig. 1.

FIG. 1: CHEMICAL STRUCTURE OF LORNOXICAM

Thiocolchicoside ²³ (Muscoril, Myoril and Neoflax) is a muscle relaxant with anti-inflammatory and analgesic effects. It acts as a competitive GABAA receptor antagonist and also glycine receptor antagonist with similar potency and nicotinic acetylcholine receptors to a much lesser extent. It has powerful convulsant activity and should not be used in seizure-prone individuals. Thiocolchicoside ²⁴ is a semi-synthetic derivative of the colchicine, a natural anti-inflammatory glycoside which originates from the flower seeds of Superba gloriosa. It is a muscle relaxant with anti-inflammatory and analgesic effects. It has potent convulsant activity ²⁵ and should not be administered to individuals prone to seizures. The IUPAC Name of Thiocolchicoside is N-[(10S)-3,4-dimethoxy- 14- (methylsulfanyl)- 13-oxo- 5-{[(2S, 3R,4S,5S,6R)-3, 4, 5-trihydroxy-6-(hydroxymethyl) oxan-2-yl] oxy}tricyclo[9.5.0.0²,⁷]hexadeca-1(16), 2, 4, 6, 11, 14-hexaen-10-yl]acetamide and the chemical formula ²⁵ is C₂₇H₃₃NO₁₀S. The chemical structure of thiocolchicoside is in Fig. 2.

FIG. 2: CHEMICAL STRUCTURE OF THIOCOLCHI-COSIDE

Literature survey ^27-30 reveals that several analytical methods have been reported for Lornoxicam and Thiocolchicoside individually in bulk and in pharmaceutical dosage forms ²⁶. Few analytical methods using spectrophotometry, HPLC and LC-MS have been reported for the simultaneous determination of Lornoxicam and Thiocolchicoside in combined dosage forms. The objective of the present study was to develop and validate a simple, accurate and precise HPLC method for simultaneous determination of Lornoxicam and Thiocolchicoside in bulk and in pharmaceutical dosage forms.

MATERIALS AND METHODS: Lornoxicam (Pure), Thiocolchicoside (Pure) gift sample procured from Sura Labs, Dilsukhnagar, Hyderabad, Water, Methanol, Acetonitrile all are HPLC grade obtained from Merck, Orthophosphoric acid, Glacial acetic acid analytical grade obtained from Merck.

Instrumentation HPLC WATERS Alliance 2695 separation module, Software: Empower 2, 996 PDA Detector.

HPLC Method Development:

Trails:

Preparation of Standard Solution: Accurately weigh and transfer 10 mg of Lornoxicam and Thiocolchicoside working standard into a 10ml of clean, dry volumetric flasks, add about 7ml of methanol, and sonicate to dissolve and remove of air completely and make volume up to the mark with the same methanol.

Further pipette 0.2ml of the above Lornoxicam and 0.3ml of the Thiocolchicoside stock solutions into a 10ml volumetric flask and dilute up to the mark with methanol.

Procedure: Inject the samples by changing the chromatographic conditions ¹ and record the chromatograms; note the conditions of proper peak elution for performing validation parameters as per ICH guidelines ^{11, 12}.

Mobile Phase Optimization: ² Initially, the mobile phase tried was Methanol: Water and Water: Acetonitrile and Methanol: Phosphate Buffer: ACN with varying proportions. Finally, the mobile phase was optimized to Methanol: Phosphate Bufferin proportion 28:72 (pH-3.8) v/v, respectively.

Optimization of Column: The method was performed with various columns like the C18 column, Symmetry, and Zodiac column. Symmetry (C18) (150mm × 4.6mm, 5µm) Column was found to be ideal as it gave good peak shape and resolution³ at 1ml/min flow.

Optimized Chromatographic Conditions:

TABLE 1: OPTIMIZED CHROMATOGRAPHIC CONDITIONS

Method Validation:

Preparation of Buffer and Mobile Phase:

Preparation of Potassium Dihydrogen Phosphate (KH₂PO₄) Buffer (pH-3.8): Dissolve 6.8043 of potassium dihydrogen phosphate in 1000 ml HPLC water and adjust the pH 3.8 with diluted orthophosphoric acid. Filter and sonicate the solution by vacuum filtration and ultrasonication.

Preparation of Mobile Phase: Accurately measured 280 ml (28%) of Methanol, 720 ml of Phosphate buffer (72%) were mixed and degassed in digital ultra sonicator for 15 minutes and then filtered through 0.45 µ filter under vacuum filtration ⁵.

Diluent Preparation: The Mobile phase was used as the diluent.

Method Validation Parameters:

System Suitability: ^{6, 7} Accurately weigh and transfer 10 mg of Lornoxicam and 10mg of Thiocolchicoside working standard into a 10ml of clean, dry volumetric flasks add about 7mL of diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent (stock solution). Further pipette 0.2ml of the above Lornoxicam and 0.3ml of the Thiocolchicoside stock solutions into a 10ml volumetric flask and dilute up to the mark with diluent.

Procedure: The standard solution was injected for five times and measured the area for all five injections in HPLC. The %RSD for the area of five replicate injections was found to be within the specified limits.

Specificity Study of Drug: ⁸

Preparation of Standard Solution: Accurately weigh and transfer 10mg of Lornoxicam and 10mg of Thiocolchicoside working standard into a 10ml of clean, dry volumetric flasks add about 7mL of Diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent (stock solution). Further pipette 0.2ml of the above Lornoxicam and 0.3ml of the Thiocolchicoside stock solutions into a 10ml volumetric flask and dilute up to the mark with diluent.

Preparation of Sample Solution: Take average weight of one Tablet and crush in a mortar by using a pestle and weight 10 mg equivalent weight ³ of Lornoxicam and Thiocolchicoside sample into a 10mL clean, dry volumetric flask and add about 7mL of diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. Further pipette 0.2ml of the sample solution from the above stock solutions into a 10ml volumetric flask and dilute up to the mark with diluents. The mean and percentage relative standard deviation were calculated from the peak areas.

Procedure: Inject the three replicate injections of standard and sample solutions ⁹ and calculate the assay by using the formula:

% Assay = Sample area / Standard area × Weight of standard / Dilution of standard × Dilution of sample / Weight of sample × Purity / 100 × Weight of tablet / Label claim

Preparation of Drugsolutions for Linearity: Accurately weigh and transfer 10 mg of Lornoxicam & 10mg of Thiocolchicoside working standard into a 10ml of clean, dry volumetric flasks add about 7mL of diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent (stock solution).

Preparation of Level – I (10ppm of Lornoxicam & 10ppm of Thiocolchicoside): Pipette out 0.1ml of Lornoxicam and 0.1ml of Thiocolchicoside stock solutions was take in a 10ml of volumetric flask dilute up to the mark with diluent.

Preparation of Level – II (15ppm of Lornoxicam & 20ppm of Thiocolchicoside): Pipette out 0.15ml of Lornoxicam and 0.2ml of Thiocolchicoside stock solutions was take in a 10ml of volumetric flask dilute up to the mark with diluent.

Preparation of Level – III (20ppm of Lornoxicam & 30ppm of Thiocolchicoside): Pipette out 0.2ml of Lornoxicam and 0.3ml of Thiocolchicoside stock solutions was take in a 10ml of volumetric flask dilute up to the mark with diluent.

Preparation of Level – IV (25ppm of Lornoxicam & 40ppm of Thiocolchicoside): Pipette out 0.25ml of Lornoxicam and 0.4ml of Thiocolchicoside stock solutions was take in a 10ml of volumetric flask dilute up to the mark with diluent.

Preparation of Level – V (30ppm of Lornoxicam & 50ppm of Thiocolchicoside): Pipette out 0.3ml of Lornoxicam and 0.5ml of Thiocolchicoside stock solutions was take in a 10ml of volumetric flask dilute up to the mark with diluent.

Procedure: Inject each level into the chromatographic system and measure the peak area. Plot a graph of peak area versus concentration (on X-axis concentration and on Y-axis Peak area) and calculate the correlation coefficient ¹⁰.

Precision:

Repeatability:

Preparation of Lornoxicam and Thiocolchico-side Product Solution for Precision: Accurately weigh and transfer 10 mg of Lornoxicam and 10mg of Thiocolchicoside working standard into a 10ml of clean, dry volumetric flasks add about 7mL of diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent (stock solution).

Further pipette 0.2ml of the above Lornoxicam and 0.3ml of the Thiocolchicoside stock solutions into a 10ml volumetric flask and dilute up to the mark with diluent.

The standard solution was injected for five times and measured the area for all five injections in HPLC ⁷. The %RSD for the area of five replicate injections was found to be within the specified limits.

Intermediate Precision: To evaluate the intermediate precision ^{13, 14} (also known as Ruggedness) of the method, Precision was performed on different days by maintaining same conditions.

Procedure:

Day 1: The standard solution was injected for three times and measured the area for all three injections in HPLC. The %RSD for the area of three replicate injections was found to be within the specified limits.

Day 2: The standard solution was injected three times and measured the area for all three injections in HPLC. The % RSD for the area of three replicate injections was found to be within the specified limits.

Accuracy: ^{15, 16}

For the preparation of 50% Standard Stock Solution: Accurately weigh and transfer 10 mg of Lornoxicamand 10mg of Thiocolchicoside working standard into a 10ml of clean, dry volumetric flasks add about 7mL of diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent (stock solution).

Further pipette 0.1ml of the above Lornoxicam and 0.15ml of the Thiocolchicoside stock solutions into a 10ml volumetric flask and dilute up to the mark with diluent.

For Preparation of 100% Standard Stock Solution: Accurately weigh and transfer 10 mg of Lornoxicam and 10mg of Thiocolchicoside working standard into a 10ml of clean, dry volumetric flasks add about 7mL of diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent (stock solution). Further pipette 0.2ml of the above Lornoxicam and 0.3ml of the Thiocolchicoside stock solutions into a 10ml volumetric flask and dilute up to the mark with diluent.

For Preparation of 150% Standard Stock Solution: Accurately weigh and transfer 10 mg of Lornoxicam and 10mg of Thiocolchicoside working standard into a 10ml of clean, dry volumetric flasks add about 7mL of diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent (stock solution). Further pipette 0.3ml of Lornoxicam and 0.45ml of Thiocolchicoside from the above stock solutions into a 10ml volumetric flask and dilute up to the mark with diluents.

Procedure: Inject the three replicate injections of individual concentrations (50%, 100%, and 150%) were made under the optimized conditions. Recorded the chromatograms and measured the peak responses. Calculate the amount found and amount added for Lornoxicam and Thiocolchi-coside and calculate the individual recovery and mean recovery values.

Robustness: ^{17, 18} The analysis was performed in different conditions to find the variability of test results. The following conditions are checked for variation of results.

For Preparation of Standard Solution: Accurately weigh and transfer 10 mg of Lornoxicam and 10mg of Thiocolchicoside working standard into a 10ml of clean, dry volumetric flasks add about 7mL of diluents and sonicate to dissolve it completely and make volume up to the mark with the same solvent (stock solution).

Further pipette 0.2ml of the above Lornoxicam and 0.3ml of the Thiocolchicoside stock solutions into a 10ml volumetric flask and dilute up to the mark with diluent.

Effect of Variation of Flow Conditions: The sample was analyzed at 0.9 ml/min, and 1.1 ml/min instead of 1ml/min; the remaining conditions ¹⁹ are the same. 20µl of the above sample was injected, and chromatograms were recorded.

Effect of Variation of Mobile Phase Organic Composition: The sample was analyzed by variation of the mobile phase, i.e., Methanol: Phosphate Buffer was taken in the ratio and 33:64, 23:77 instead (28:72), remaining conditions are same. 20µl of the above sample was injected, and chromatograms were recorded.

RESULTS AND DISCUSSION: Method Development:

METHOD VALIDATION: The proposed method was subjected to validation for various parameters like linearity and range, precision, accuracy, and robustness in accordance with International Conference on Harmonization Guidelines.

Linearity: The linearity of an analytical method is its ability to elicit test results that are directly, or by a well-defined mathematical transformation, proportional to the concentration of an analyte in samples within a given range. Weigh accurately 10mg of Lornoxicam and 10mg of Thiocolchicoside in 10 ml of volumetric flask and dissolve in 7ml of mobile phase and make up the volume with mobile phase. This solution contains 10-30 μg/ml of Lornoxicam and 10-50 μg/ml of Thiocolchicoside.

Acceptance Criteria: The correlation coefficient should be not less than 0.999. The linearity data and respective chromatograms for Lornoxicam and Thiocolchicoside are as follows.

TABLE 2: CALIBRATION DATA OF LORNOXICAM

FIG. 10: CALIBRATION CURVE OF LORNOXICAM

TABLE 3: CALIBRATION DATA OF THIOCOLCHICOSIDE

FIG. 11: CALIBRATION CURVE OF THIOCOLCHICOSIDE

Precision: Method precision, also called repeatability/Intraday precision indicates whether a method gives consistent results for a single batch. Method precision was demonstrated by preparing five test solutions at 100% concentration as per the test procedure & recording the chromatograms of five test solutions. The % RSD of peak areas of five samples was calculated. The method precision was performed on Lornoxicam and Thiocolchico-side.

TABLE 4: RESULTS OF REPEATABILITY FOR LORNOXICAM

TАBLE 5: RESULTS ОF REPEATABILITY FОR THIOCOLCHICOSIDE

Intermediate Precision or Ruggedness: The ruggedness of the method was verified by analyzing three samples of the same batch used for method precision as per the proposed method by the different analysts. The repeatability of sample applications and measurement of peak area was expressed in terms of %RSD since their %RSD is <2.0%, and hence, the developed method was found to be precise. Data obtained from intermediate are summarized in Table 6, 7 and 8, 9.

Day 1:

TABLE 6: RESULTS ОF INTERMEDIATE PRECISION DАY1 FОR LORNOXICAM

TABLE 7: RESULTS ОF INTERMEDIATE PRECISION DАY 1 FОR THIOCOLCHICOSIDE

Day 2:

TАBLE 8: RESULTS ОF INTERMEDIATE PRECISION DAY 2 FОR LORNOXICAM

TABLE 9: RESULTS ОF INTERMEDIATE PRECISION DAY 2 FОR THIOCOLCHICOSIDE

Accuracy: The accuracy of the method was determined by recovery experiments. The recovery studies were carried out at three levels of 50%, 100%, and 150% and the percentage recovery was calculated and presented in Tables 10 and 11. Recovery was within the range of 98%-102%, which indicates the accuracy of the method.

Аccurаcy 50%:

Аccurаcy 100%:

Аccurаcy 150%:

TABLE 10: THE ACCURACY RESULTS FОR LORNOXICAM

TABLE 11: THE ACCURACY RESULTS FОR THIOCOLCHICOSIDE

LOD and LOQ: The LOD and LOQ were calculated using the following equation as per ICH guidelines:

LOD = 3.3 × σ / s

LOS = 10 × σ / s

Where σ is the standard deviation of ?-intercepts of regression lines and ? is the slope of the calibration curve.

The results of LOD and LOQ are summarized in Table 12.

TABLE 12: LOD AND LOQ VALUES OF LORNOXICAM AND THIOCOLCHICOSIDE

Robustness: The robustness of the method was studied by deliberately changing the experimental conditions like flow rate and percentage of mobile phase ratio. The study was carried out by changing 5% of the mobile phase ratio and 0.1 mL/min of flow rate.

Vаriаtiоn in Flow:

Vаriаtiоn of Mobile Phase Organic Composition:

TABLE 13: RESULTS FОR ROBUSTNESS–LORNOXICAM

TABLE 14: RESULTS FОR ROBUSTNESS–THIOCOLCHICOSIDE

System Suitability: A system suitability test was an integral part of the method development to verify that the system is adequate for the analysis of Lornoxicam and Thiocolchicoside to be performed. System suitability test of the chromatography system was performed before each validation run. Five replicate injections of a system suitability standard and one injection of a check standard were made. Area, retention time (RT), tailing factor, asymmetry factor, and theoretical plates for the five suitability injections were determined.

CONCLUSION: In the present investigation, a simple, sensitive, precise, and accurate RP-HPLC method was developed for the quantitative estimation of Lornoxicam and Thiocolchicoside in bulk drug and pharmaceutical dosage forms. Lornoxicam was found to be soluble in the organic solvents ethanol, DMSO, and dimethylformamide (DMF) and slightly soluble in water and Acetonitrile. Thiocolchicoside was found to be soluble in water, methanol, 0.1N HCl, 0.1N NaOH. Methanol: Phosphate Buffer (pH-3.8) (28:72% v/v) was chosen as the mobile phase. The solvent system used in this method was economical. The %RSD values were within 2, and the method was found to be precise. The results expressed in tables for the RP-HPLC method was promising. The RP-HPLC method is more sensitive, accurate, and precise compared to the spectrophotometric methods. This method can be used for the routine determination of Lornoxicam and Thiocolchicoside in bulk drug and in pharmaceutical dosage forms.

ACKNOWLEDGEMENT: The author is thankful to Sura Labs, Dilsukhnagar, for providing the necessary facilities to carry out this work.

SOURCE OF SUPPORT: Nil

CONFLICTS OF INTEREST: The author declares no conflict of interest.

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    How to cite this article:

    Jena DK: Analytical method development and validation for the simultaneous estimation of lornoxicam and thiocolchicoside in bulk form and marketed pharmaceutical dosage form by using RP-HPLC. Int J Pharm Sci & Res 2021; 12(2): 1020-32. doi: 10.13040/IJPSR.0975-8232.12(2).1020-32.

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