Simple Gas Chromatographic Method Research Articles

Simultaneous Determination of General Active Ingredients in Topical Formulations by Gas Chromatography with Flame Ionization Detection (GC-FID).

A simple, fast, precision and sensitive gas chromatographic method was developed for determination of camphor, L-menthol, methyl salicylate, salicylic acid, benzyl nicotinate and nonivamide in topical formulations. The possibility of determination low-boiling components and high-boiling components by a single analytical method was shown. The method was optimized by evaluating the effect of a large number of chromatographic parameters and sample preparation stages. It was validated according to the International Council on Harmonization guidelines. Limit of quantification of active components were achieved from 0.7μg/mL for benzyl nicotinate and L-menthol to 41μg/mL for salicylic acid. High repeatability and good recovery values were demonstrated. The practical application of the proposed method was shown for ointments and cream.

Validated method for the determination of free volatile carboxylic acids in cheese and bacterial cultures by GC-FID after esterification in aqueous extract and headspace injection

A simple, rapid, sensitive and robust gas chromatographic method was developed for the simultaneous determination of free volatile carboxylic acids (FVCA) in cheese and bacterial cultures. The target analytes were extracted and converted directly from the aqueous phase to their ethyl esters using headspace. The lower detection limits for the volatile carboxylic acids in the cheese samples were less than 0.3 and less than 0.6 µmol kg−1 in the bacterial culture samples. The lower limits of quantitation in cheese were better than 0.001 mmol kg−1 for all analytes. The upper limits of quantitation varied from 39 to 136 mmol kg−1 in cheese and 78 to 272 mmol kg−1 in bacterial cultures depending on the analyte. The Horwitz ratio showed good precision for all analytes (less than 0.77). The proposed method is suitable for the determination of target metabolites directly from aqueous extracts and can also be validated for other matrices.

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A rapid and simple Gas Chromatographic (GC) method has been developed for the simultaneous quantification of eugenol, thymoquinone and β-careophyllene in individual and mixed spice samples. Analysis was performed using capillary column (30 m x 0.25 mm x 0.25 µm) by using Flame ionization detector. Mixed spices were extracted by methanol. The calibration plot was linear over the range studied (2-500 ng for each eugenol, thymoquinone and β-careophyllene standards) with a correlation of 0.99 for eugenol, thymoquinone and β-careophyllene. The method was also validated for the linearity, range, precision, recovery and detection limits. Thus, the method is suitable for routine analysis of eugenol, thymoquinone and β-careophyllene in mixed spices.

Method Development and Validation for the Determination of Residual Solvents in Racecadotril by Gas Chromatography

Background: The presence of residual solvents in pharmaceuticals can be a potential risk factor to human health because of its toxicity. Gas chromatography is used for its excellent separation abilities and lower limit of detection. Racecadotril, an antidiarrheal drug, is reported having four residual solvents, namely n-Hexane, isopropyl alcohol, toluene, and dimethylformamide. Estimation of the amount of these solvents in active pharmaceutical ingredients to ensure that they are within the permissible limits as per ICH guidelines is necessary. Objective: To develop and validate a new, simple, and sensitive gas chromatographic method for simultaneous determination of n-Hexane, isopropyl alcohol, toluene, and dimethylformamide in racecadotril. Methods: The residual solvents of racecadotril were estimated using a gas chromatographic method by direct injection using FID as a detector. This method employed a 30-meter long DB-FFAP nitroterephthalic-acid-modified polyethene glycol column with 0.53 mm in inner diameter and 1 μm film thickness. The separation was achieved using nitrogen as the carrier gas at a flow rate of 2.8 mL/min using a split ratio of 1:10. Results: The peak shape for all the residual solvents from racecadotril was symmetric with excellent resolution eluting at reasonable retention time. The limit of detection of n-Hexane, isopropyl alcohol, Toluene, and dimethylformamide was found to be 6, 27, 14, and 42 ppm, respectively. The developed method exhibited excellent linearity for each residual solvents in the range studied. Conclusion: The developed gas chromatographic method is simple, specific, precise, accurate, and sensitive. Hence, the method can be successfully used in in the pharmaceutical companies and research laboratories for simultaneous determination of residual solvents in racecadotril active pharmaceutical ingredients.

Determination of residual solvents in paclitaxel by headspace gas chromatography

BackgroundA simple and sensitive gas chromatographic method was developed and validated for the simultaneous determination of methanol, ethanol, acetone, isopropyl alcohol, dichloromethane, N-hexane, ethyl acetate, tetrahydrofuran, and N,N-diisopropyl ethyl amine in Paclitaxel. A chromatographic separation was done on DB-624 column, 30 m length × 0.53 mm ID, and film thickness 3 μm, using a flame ionization detector (FID) with gradient column oven temperature program. The injection was carried out in split mode, with a split ratio of 5:1. A mixture of N-methyl-2-pyrrolidinone (contains 1% piperazine) and water in the ratio of 80:20 (v/v) was selected as a diluent to obtain good sensitivity along with the recovery.ResultsThe developed gas chromatographic method offers symmetric peak shape, good resolution of more than 2.0 between the solvent peaks, and the relative standard deviation for replicate injections of all the solvents were found to be not more than 15.0% with reasonable retention time for all the solvents. The limit of detection for methanol, ethanol, acetone, isopropyl alcohol, dichloromethane, N-hexane, ethyl acetate, tetrahydrofuran, and N,N-diisopropyl ethyl amine was found to be 304.69 ppm, 497.98 ppm, 498.99 ppm, 504.49 ppm, 61.81 ppm, 30.07 ppm, 505 ppm, 73.05 ppm, and 2.09 ppm, respectively. Limit of quantitation of methanol, ethanol, acetone, isopropyl alcohol, dichloromethane, N-hexane, ethyl acetate, tetrahydrofuran, and N,N-diisopropyl ethyl amine was found to be 89.62 ppm, 146.47 ppm, 146.76 ppm, 148.38 ppm, 18.18 ppm, 8.84 ppm, 148.53 ppm, 21.49 ppm, and 0.62 ppm, respectively. Precision was found to be satisfactory. Linear in the range of LOQ to 150% level for all the solvents, and accuracy along with robustness, is performed, and acceptable results were obtained.ConclusionThe proposed method was demonstrated to be simple, sensitive, specific, linear, precise, accurate, and robust, hence can be used to determine the residual organic solvents in Paclitaxel drug substance and drug product.

Development and Validation of a Headspace Gas Chromatographic (HS-GC) Method for Determination of Residual Solvents in Nitazoxanide API

Controlling residual solvents in the drug substances or active pharmaceutical ingredients (API) is mandatory to the specified limits as per the International Conference on Harmonisation (ICH) Q3C guidelines. Residual solvents in pharmaceuticals are mostly determined by Gas Chromatography with Headspace. A simple and sensitive headspace gas chromatographic (HS-GC) method has been developed for the determination of Acetone, Dichloromethane and Cyclohexane in Nitazoxanide API. The separation of analytes was achieved with DB – 624 (30 m length, 0.53 mm inner diameter and 3.0 μm in film thickness) capillary column. Dimethyl formamide was used as a diluent. Nitrogen was used as carrier gas with 3.0 mL/minutes and Flame ionisation detector (FID) for detecting analytes. The oven temperature was set at 60°C for 5 minutes at initial and programmed at a rate of 20°C per minute to a final temperature of 240°C for 2 minutes. Run time was 16 minutes, and total GC cycle time was 25 minutes. The spilt ratio used as 1:20 to get optimum peak response. The developed method was validated as per the ICH guidelines for specificity, accuracy, precision, linearity, range, the limit of detection, the limit of quantification and robustness. The results of validation were indicated no interference, good recoveries, precise, linear, rugged and robust method, suitable for the determination of residual solvents in Nitazoxanide API for research and routine quality control laboratory.

A NOVEL VALIDATED HEADSPACE GAS CHROMATOGRAPHY METHOD FOR THE SIMULTANEOUS QUANTIFICATION OF THREE ORGANIC VOLATILE IMPURITIES IN ENROFLOXACIN PURE AND PHARMACEUTICAL DOSAGE FORMS

Objective: This article describes a novel, simple, and rapid gas chromatographic method for quantification of three organic volatile impurities (OVIs) present in enrofloxacin and its pharmaceutical dosage forms.
 Methods: As stationary phase using ZB-624 30 m×0.53 mm, 3.0 μ column with flame ionized detector at 250°C. The injector temperature is maintained at 180°C. The nitrogen gas was used as a carrier gas with a flow rate of 4.0 mL/min. The method involved a thermal gradient elution. The total run time is 21.14 min.
 Results: The retention time of three OVIs taken individually and in spiked standard solutions were determined. The retention times are 2.30 min for methanol, 7.07 min for 1-butanol, and 8.48 min for toluene, respectively. The % relative standard deviation for six injections should be not more than 10%. The % recovery ranges from 85 to 115%. The correlation coefficient (r2) for linearity is not <0.99. The limit of quantification was found to be 260 ppm for methanol, 101 ppm for 1-butanol, and 56 ppm for toluene. Furthermore, verified precision, ruggedness, robustness, solution stability, and pharmaceutical analysis.
 Conclusion: All the obtained results are found within the acceptable limits. The proposed method has been successfully applied for the quantification of OVIs present in enrofloxacin pure and its pharmaceutical dosage forms.

Cyclodextrin-based Gas Chromatography and GC/MS methods for determination of chiral pair constituents in mint essential oils

ABSTRACT The genus Mentha has been known as culinary source of minty note. Besides, the essential oil imparts value to consumer products like confectionery, cosmetics, and pharmaceutical preparations. A simple and sensitive enantioselective gas chromatographic method is described for the quantitative analysis of terpenoid enantiomers in essential oils using different cyclodextrin coated stationary phases. Limonene, menthone, menthol, and menthyl acetate were identified as pure (-)-enantiomers whereas isomenthone, neomenthol, pulegone, and piperitone as pure (+)-enantiomers in menthol mint, and peppermint oils, respectively. The selectivity of each enantiomer was also demonstrated. A β-cyclodextrin phase with diethyl substitution provides baseline resolution for all target enantiomers with selectivity ranged from 1.004 to 1.050. Moreover, permethylated or diacetylated-β-cyclodextrin showed no selectivity for (+/-)-menthol enantiomers. The current study may facilitate the origin authentication studies of mint oils. Investigation of enantiomers involved in menthol biosynthesis revealed that only (-)-menthol is present in the essential oils of Mentha species.

Aliphatic Hydrocarbons Extraction from Oily Sludge using Kerosene

The National Petroleum Refinery of Cameroon is a crude oil refining company that generates large quantity of oily sludge. The extraction, identification and quantification of aliphatic hydrocarbons that can be recovered from oily sludge were assessed. The extraction was conducted following an experimental design and the distribution of the aliphatic hydrocarbons of the sludge was realized by using a simple gas chromatographic method. The extraction results showed that all the factors have positive effect on the extraction. It was also found that the highest concentration of 127992 ppm was obtained under experimental conditions of Ratio = 6:1, Temperature = 40°C, Contact time = 90 min.

Aliphatic Hydrocarbons Extraction from Oily Sludge using Kerosene

The National Petroleum Refinery of Cameroon is a crude oil refining company that generates large quantity of oily sludge. The extraction, identification and quantification of aliphatic hydrocarbons that can be recovered from oily sludge were assessed. The extraction was conducted following an experimental design and the distribution of the aliphatic hydrocarbons of the sludge was realized by using a simple gas chromatographic method. The extraction results showed that all the factors have positive effect on the extraction. It was also found that the highest concentration of 127992 ppm was obtained under experimental conditions of Ratio = 6:1, Temperature = 40°C, Contact time = 90 min.

Gas Chromatographic and Spectrophotometric Determination of Diclofenac Sodium, Ibuprofen, and Mefenamic Acid in Urine and Blood Samples.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of acute to chronic pain. A simple, fast, and reliable gas chromatographic (GC) method with flame ionization detection has been developed for the determination of NSAIDs such as diclofenac sodium, ibuprofen, and mefenamic acid after derivatization with ethyl chloroformate. The GC conditions were optimized as elution from a DB-1 column (30 mx0.32 mm id) at column temperature 150 °C for 3 min, followed by a heating rate of 20 °C/min up to 280 °C and a hold time of 5 min. The nitrogen flow rate was 2.5 mL/min. For spectrophotometric studies, the absorbance was measured against methanol at a wavelength of 200-500 nm. The calibration curves were linear within 2-10 μg/mL with limits of detection of 0.4-0.6 μg/mL of each drug. The derivatization elution, separation, and quantitation were repeatable (n=3) with relative standard deviation (RSD) within 3.9%. The method was applied for the analysis of the drugs from pharmaceutical formulations and the results of the analysis agreed with labeled values with RSDs within 0.5-3.9%. The results were also confirmed by standard addition method. The percent recovery was calculated with spiked deproteinized human blood serum and urine samples and % recovery of the drugs was obtained within 96-98% with RSDs within 3.1%. The validated method proved its ability for the assay of NSAIDs in bulk and dosage form in a short analysis time. The method was also useful for the analysis of biological samples.

Determination of Ethanol Content in Kombucha Using Headspace Gas Chromatography with Mass Spectrometry Detection: Single-Laboratory Validation.

BackgroundKombucha is a fermented beverage made with tea, sugar, and a symbiotic colony of bacteria and yeast that is usually marketed as a non-alcoholic beverage. Products must contain <0.5% and <1.1% alcohol by volume in the United States and Canada respectively to be classified as non-alcoholic products. Prior studies have found that Kombucha beverages can become very acidic and may contain levels of alcohol above 1% which can be a potential health risk to children and the developing fetus during pregnancy.ObjectiveGiven the public safety concerns and legal requirements associated with the level of alcohol within Kombucha beverages, there is a need for accurate and reliable methods. Herein we describe the validation of a sensitive, rapid, and simple Headspace Gas Chromatographic method with mass spectrometric detection for determining ethanol in Kombucha.MethodsMethod performance characteristics measured included linearity, accuracy, precision, limit of detection (LOD), and limit of quantification (LOQ) as per AOAC International guideline Appendix K Part 1. Performance was evaluated against the AOAC Standard Method Performance Requirements 2016.001 for determination of ethanol in Kombucha.ResultsThe linear dynamic range for this method was confirmed over the range of 0.025 to 2.47% ABV. The LOD and LOQ were determined to be 0.0002% and 0.002% ABV, respectively. With a spike recovery of 102% for accuracy and precision of RSDr ≤ 4% the method met the SMPR requirements within the analytical range.ConclusionsThe results of this validation study demonstrated the method is fit for the purpose of quantifying ethanol in Kombucha and is suitable for rapid and easy integration by laboratories to ensure that regulatory requirements are met.

A Rapid and Simple Gas Chromatographic Method for Direct Determination of Safrole in Soft Drinks

A simple and rapid method was developed to determine the safrole and isosafrole in soft drink using a megapore semi-polar column (CP-SIL 8CB, 30 m × 0.53 mm, 1.5 μm) with direct injection gas chromatography. Direct quantitative analysis of safrole and isosafrole in soft drinks was carried out without any sample pretreatment procedure. The water soluble compound 1,4-dihydroxybenzene (DHB) was used as the internal standard. The detection limit for safrole and isosafrole was 0.25 μ/mL. A recovery study was performed using one of the soft drinks by spiking 1 mL with safrole and isosafrole at 5.0 and 10.0 μg, respectively. The recovery was found in the range of 98-108% with coefficients of variation being less than 8.7%. Twenty-five commercial soft drinks were analyzed by the current method, and results indicated that 20 out of 25 soft drink samples contained safrole andlor isosafrole, and the amount of safrole was 3-5 fold over the regulated amount, 1 μ/mL.

A rapid and simple gas chromatographic method for direct determination of nicotinamide in commercial vitamins and tonic drinks

A simple and rapid method was developed to determine the presence of nicotinamide in tonic drinks using the megapore semi-polar column (CP-Sil 8 CB, 30 m x 0.53 mm) with direct injection gas chromatography. Direct quantitative analysis of nicotinamide in amino acid drinks, vitamin drinks, essence of chicken and tonic drinks was carried out without any sample pretreatment procedure. Water soluble compound 1,9-nonanediol was used as an internal standard. The detection limit for nicotinamide was 2-5 μg/mL. Recovery studies were performed using 1 mL of vitamin drink and tonic drink, each spiked with nicotinamide at 105.5, and 211.0 μg, respectively. The recoveries were found in the range of 94-99% and 93-108%, respectively. The coefficients of variation being less than 9.8%. Forty-two commercial amino acid drinks, vitamin drinks, essences of chicken and tonic drinks were analyzed with this method. The nicotinamide contents were found as: 131-246, 24-112, 0-134 and 0-263 μg/mL, respectively. These results indicated that, one bottle each of the amino acid drink, vitamin drink, essences of chicken and tonic drink contain nicotinamide levels of: 39-74, 5-22, 0-10 and 0-21 mg/bottle. The results also showed that in 12 out of 4 of commercial amino acid and vitamin drinks, the nicotinamide exceeded the ROC's acceptable daily intake (RNDA, 14.4 mg) level; and 3 out of 10 commercial tonic drinks exceeded the ROC's RNDA level for nicotinamide.

A Simple, Specific, Mass Compatible and Validated Gas Chromatographic Method for the Estimation of Piperidine-3-amine Content in Linagliptin Finished and Stability Samples without Derivatization

A new simple, selective, highly sensitive, specific (stability indicating), robust, rugged and mass compatible gas chromatographic method and sample with direct injection-mode was developed for the quantitative determination of amino-3-piperidine (3-aminopiperidine, 3-AMP) in linagliptin. As the CAD-LC method did not proved for specificity and pre-derivitizations are challenging task for quality control (QC), a simple GC method has been developed. Compared to LC-CAD method, which is not proved for specificity and pre-column derivitization methods having the limitations to analyze the reaction monitoring in-process samples and degradation samples, the present method is selective, simple, cost effective, QC friendly, widely available GC technique with direct injection and high in sensitivity. Also this method is mass compatible, specificity proved by forced degradation, method was validated as per ICH guidelines. Mass balance was proved by analyzing the stressed samples for net degradation by HPLC and assay by HPLC methods. This GC method also provides an advantage to analyze the in-process samples to monitor the progress of the synthetic process, where the reaction monitoring samples are unpurified or non-isolated samples and contains many process related impurities (reference) and solvents, which will have interference with 3-AMP in LC-CAD and pre-column derivitization methods. This method involves simple sample preparation process and direct injection with GC-FID technique. Hence, this method can be used to analyze the finished product samples, degradation samples, stability samples and reaction monitoring samples. The method was developed with widely available GC column (diphenyl dimethyl polysiloxane as stationary phase, 30 m length, 0.53 mm internal diameter &amp; 5.0 μm thickness), helium as carries gas, FID detector set at 240 ºC, column oven starts at 200 ºC and starts increases after 2 min with 20 ºC/min and holds up to 11 min, which will ensure the column bake. The solvents used for the process were well separated from 3-AMP peak. Mass balance was reported &gt; 99%. The limit of quantification and limit of detection values for 3-AMP were 0.002% (0.4 μg/mL) and 0.007% (1.4 μg/mL) of targeted concentration (20 mg/mL), respectively. The method was precise at LOQ and at specification level with %RSD values 2.8 and 4.7, respectively. Linearity was established in the range of 0.0014 mg/mL (LOQ) to 0.045 mg/mL for 3-AMP with correlation coefficient (r2 &gt; 0.9995). The percentage recoveries were obtained between 99.9% and 104.4%. The range of the method was established from LOQ (0.0014 mg/mL) to 150% (0.045 mg/mL) of the specification targeted limit of 0.15% (0.03 mg/mL). The standard and spiked sample solutions were studied up to 2 days and are stable at room temperature. The forced degradation studies were performed by using HCl, NaOH, H2O2 thermal, UV radiation and water. A mild degradation bout 0.25% was observed in base degradation condition, which was confirmed with mass number by GC-MS analysis. Validation parameters were evaluated according to the International Conference on Harmonization (ICH) Q2 guidelines.

A simple high-throughput headspace gas chromatographic method for the determination of dissolved oxygen in aqueous samples

This paper reports on a headspace gas chromatographic method (HS-GC) for the accurate determination of dissolved oxygen (DO) in domestic and industrial wastewater. It was based on GC measurement of oxygen in the headspace gas that is released from the dissolved oxygen in a liquid sample in a closed vial. The results show that the relative standard deviation of the method in quintuplicate testing was < 2.0% and the relative differences of results were less than 4.0% for the real river water samples when compared with the widely used reference method (the electrode method); The linear range of the method is about 0–9.0 mg/L with the limit of detection of 0.30 mg/L. The present method is simple in both sample preparation and method calibration. It is particularly efficient in the batch testing mode. Overall, the method is more reliable than the current reference method when used with samples from a wide range of sources.

Assessment of compliance level of ICH guidelines for organic volatile impurities in common ayurvedic hepatic formulations.

Background Herbal medicines have been used in the treatment of liver diseases for a long time. In recent years, the use of herbal medicines for protection from other strong antibiotics as well as drugs that can damage the liver during their metabolism in liver and for treatment of liver diseases has increased all over the world. It is important to mention that a number of organic solvents are used at different stages of extraction/formulation development for these traditional preparations in industries/pharmacies. In addition, some of these solvents possess established carcinogenic properties and may enter the formulation as residual solvents. Hence as per ICH guidelines it is mandatory to keep the level of these solvents up to permissible limits. There has been a lot of hue and cry that ayurvedic formulations available in the market are not properly standardized for their quality due to lack of stringent regulations and standards from regulatory authorities. Therefore the aim of present work was to assess the compliance of ICH guidelines for level of organic volatile impurities in common marketed ayurvedic hepatic formulations. Methods In this study, 25 ayurvedic herbal formulations available as OTC product have been assessed for presence of residual solvents using gas chromatography with flame ionization detector. Results This study on 25 fast moving hepatic formulations in the market reflects that no residual solvents were detected in any of the formulations however if present were within prescribed permissible limits of ICH guidelines. The data was also subjected to statistical analysis (F-test and t-test at 95% confidence level). Conclusions Results indicate the safety of these hepatic formulations with respect to residual solvents. In addition presents a simple, linear, specific, accurate, precise and rugged gas chromatographic method for estimation of residual solvents.

A simple and efficient headspace gas chromatographic method for the determination of carboxyl groups in nanofibrillated cellulose

This paper reports on a headspace gas chromatography method (HS-GC) for efficient determination of carboxyl groups in nanofibrillated cellulose (NFC). By pretreating the NFC suspension with a strong acid cation exchange resin for 30 min, the carboxyl groups in NFC were converted to the carboxyl acids. The pretreated suspension was added into a closed headspace sample vial containing bicarbonate solution. By HS-GC measuring the CO2 produced from the reaction between carboxyl acids and bicarbonate (10 min at 60 °C), the content of carboxyl groups in NFC can be determined. The results showed that the relative standard deviation of the method in the repeatability testing was < 3.7% and the limit of quantitation was about 0.1 mmol/g. Relative differences between the HS-GC and the reference (a conductometric titration) method were within 15.0%. The present method is simple and efficient, and suitable to be used for determining the content of carboxyl groups in NFC in the related applications.

Fate of acephate and its toxic metabolite methamidophos during grape processing

This study aimed to determine the degradation of acephate and its metabolite methamidophos during different kinds of grape processing, using a simple gas-chromatographic method with a flame photometry detector. During wine processing, there was no significant effect of crushing, pressing, and clarification on the removal of acephate and methamidophos, whereas 58.9% acephate was dissipated during the fermentation step. Aging and bottling resulted in a reduction of 43.2% and 30.6% for acephate and methamidophos, respectively. The processing factors (PFs) of acephate during the wine process were <1, whereas those of methamidophos were >1. Moreover, washing with hydrogen peroxide solutions effectively reduced the levels of acephate and methamidophos present in the grape skin and pulp more than other washing solutions did (tap water, detergent solution, acetic acid solution, NaCl solution, and Na2CO3 solution). Finally, the best removal rate of acephate residue and the minimum methamidophos level was achieved at a fermentation temperature of 22.4 °C, yeast inoculum of 8.5‰, and pH of 3.8. These results suggest that the potential metabolites of parent pesticide should be paid more attention during wine processing.

Fast and simple gas chromatographic method for simultaneous estimation of camphor, menthol and methyl salicylate in analgesic ointment: application in stability study

A simple, rapid and sensitive gas chromatographic (GC) method with flame ionization detector (FID) has been developed and validated for simultaneous estimation of camphor, menthol and methyl salicylate (MS). Camphor, menthol and MS were separated at about 2.753, 3.206 and 3.995 min respectively on a capillary column with helium (3.3 ml/min) as carrier gas within 11 min run time. Noninterference of any peak with the peaks of interest confirms the selectivity of method. Derived quantitation limits (QL) were 0.847, 0.684 and 6.507 µg/ml for camphor, menthol and MS respectively. The linear relationship (R2 > 0.999) between analyte concentration vs detector response was established within a range of QL to 150% of label claim concentration for each analyte. Recovery of each analyte at 50, 100 and 150% of label claim concentration levels were obtained within 99.67–101.53% establishing high accuracy of the method. The method showed acceptable precision with low relative standard deviation or RSD (0.24–1.03%) between percent recoveries for each analyte. RSD for intermediate precision (inter day analysis, analyst variation) was less than 1%. The validated method was successfully applied for quantitative determination of camphor, menthol and MS in stability samples of an analgesic ointment produced by IKOP Sdn. Bhd., Malaysia.