Simultaneous Estimation of Ketorolac Tromethamine and Tiemonium Methylsulphate in Biofluids by a Validated UPLC Method


Indo. J. Phar. Scie. Tech. Vol. 5, No. 1, 15-25 (2018).

Mohammad Nasir Uddin*, Monir Uddin, Md. Touhidul Kabir

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Department of Chemistry, University of Chittagong

Korespondensi : (Mohammad Nasir Uddin)

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An Ultra Performance Liquid Chromatography (UPLC) method has been developed for the simul- taneous determination of ketorolac tromethamine (KTR) and tiemonium methylsulphate (TMS) in pharmaceutical and bio-samples. Calibration curves were linear with correlation coefficient 0.9997 at a studied concentration range of (0.1-10 μg mL-1) for both drugs. The method was reproducible with relative standard deviation (RSD) for intraday precision was 0.32-0.99% for KTR and 0.40-1.01% for TMS and inter day precision was 0.51-2.77% for KTR and 0.41-2.26% for TMS. The mean recovery of the intraday assay was 99.18-103.34% for KTR and 99.14-101.76% for TMS and inter-day assay at six different days was 99.39-102.04% for KTR and 98.08-103.78% for TMS. The limit of detec- tion (LOD) and limit of quantitation (LOQ) were 0.125 and 0.41 μg mL-1, respectively for KTR and 0.150 and 0.50 μg mL-1, respectively for TMS. The method was successfully validated as per the ICH guidelines can be conveniently employed for routine quality control analysis of KTR and TMS in pharmaceutical formulations and biological samples without any interference.
Key words : Ketorolac tromethamine, tiemonium methylsulphate, UPLC, pharmaceutical and biosamples.


Ketorolac tromethamine (Figure 1: KTR), 2-Amino-2-(hydroxyl methyl) pro- pane-1, 3-diol (1RS)-5-benzoyl‐2,3-dihy- dro-1H-pyrrolizine-1-carboxylate, is a non- steroidal anti-inflammatory drug (NSAID). It is a white crystalline powder and freely soluble in water, methanol and ethanol. It is a member of the heterocyclic acetic acid de- rivative family. It is used as an analgesic with an efficacy close to that of the opioid family1. It is also a potent antipyretic and anti-inflammatory. It is mainly used for the short term treatment of post-operative pain as it is highly selective for the cylcooxygenase (COX-1) enzyme2.

KTR is metabolized through hydroxy- lation in the liver to form p-hydroxy ketorolac and its metabolites are primarily excreted in the urine (91%), and the rest is eliminated in the feces. Other adverse effects are similar to the ones associated with other NSAIDs. The most serious risks associated with ketorolac are those associated with other NSAIDs, i.e. gastrointestinal ulcers, bleeding and perfo- ration; renal events ranging from interstitial nephritis to complete kidney failure; hemor- rhage, and hypersensitivity. Ketorolac also causes rise in serum transaminase levels. Allergic reactions (anaphylactoid reactions, asthma, bronchospasm, Stevens–Johnson syndrome, and toxic epidermal necrolysis) have been reported. Fluid retention and ede- ma have been reported with the use of ke- torolac and it should therefore be used with caution in patients with cardiac decompensa- tion, hypertension or similar conditions3,4.

Tiemonium methylsulphate (TMS) (Figure 2) 4-[3-Hydroxy-3-phenyl-3-(2-thie- nyl) propyl]-4-methyl morpholinium methyl- sulphate is quaternary ammonium antimus- carinics with peripheral effects similar to

those of atropine and are used in the relief of visceral spasms. It reduces muscle spasms of the intestine, biliary system, uterus & urinary bladder. It is indicated for the pain in gas- trointestinal & biliary disease in the urology and gynecology such as gastroenteritis, diar- rhea, dysentery, biliary colic, enterocolitis, cholecystytis, colonopathyes, mild cystitis, & spasmodic dysmenorrhea5.

Adverse drug interactions in the concomitant use of ketorolac tromethamine and tiemonium methylsulphate are not well docu- mented. This adverse interaction may effect on their plasma level. Their simultaneous determination in pharmaceutical formulation and biosamples is immense important. It was found that though individually these drugs have been analyzed by many methods6,7,8,9,10 no one is available for their simultaneous es- timation in a single run. Attempts have been made to develop new methods the estimation of KTR and TMS in pharmaceutical and bi- osamples. Chromatographic techniqus such as high-performance liquid chromatography (HPLC) with with diode array detection11,12 and gas chromatography (GC) with electron capture detection have been used13. GC-MS methods using either electron impact (EI) or chemical ionization (CI) have been reported, but these procedures still require a tedious derivatization step prior to final analysis. However, liquid chromatography methods do not require a derivatization step.



In this paper UPLC method for simul- taneous determination of ketorolac tromith- amine and tiemoniummethylsulphate has been reported. The proposed method is opti- mized and validated according to ICH guide- lines14. This simple, accurate, precise and sensitive method can also be used for the rou- tine analysis of both drugs in mixture without time consuming.

Materials and Methods


HPLC-grade methanol was supplied by Sigma-Aldrich (Germany), Acetonitrile was supplied by Scharlau (Scharlab S.L, Spain) and sodium dihydrogen phosphate was sup- plied by Applichem GmbH (Germany). Wa- ter used throughout the study was purified by the reverse osmosis method to gain high- purity water with a Milli-Q water purification system from Millipore (Millipore, Bedford, MA, USA). Purity of reference compounds was not less than 98%.

Pharmaceutical formulations commercially available in Bangladesh were analysed to check the applicability of the method: Torax (10 mg) tablet by Square, Rolac (10 mg) tablet by Renata, Etorac (10 mg) tablet by Incepta, Zidolac (10 mg) tablet by Beximco, ketonic (10 mg) tablet by SK+F, Torax (30 mg) injection by Square, Rolac (30 mg) injection by Renata, Norvis (50 mg) tablet by Square, Visceralgin (50 mg) tablet by Nuvista, Timozin (50 mg) tablet by Incepta, Visrul (50 mg) tablet by Opsonin, Algin (50 mg) tablet by Renata, Visceralgin (5 mg) Injection by Nuvista, align (5 mg) Injection by Renata, align (10 mg) syrup by renata, visrul (10mg) syrup by opsonin. Biological samples, blood or urine (4 mL) were collected in bottles from the male patient under treatment with Algin after 1 hour of injection administration in Chittagong medical college and hospital, Bangladesh.

Preparation of Standards

Stock solutions of tiemonium methyl- sulphate were prepared at concentration level 100 μg mL-1 by dissolving an appropriate amount of each compound in ethanol and were stored at 4OC, protected from light and used within 3 months. The stock solutions of drugs were further serially diluted daily be- fore analysis with ethanol to make interim mixture solutions (controlled solution) at concentrations of 1, 3, 5, 7, 10 μg mL-1 for the compound. Buffer: 5 mM aqueous solu- tion of dihydrogen sodium phosphate buffer was prepared by mixing appropriate weight in Milli Q water and filtered before use.

2.3. Sample Preparation

2.3.1.Pharmaceutical Samples

Twenty tablets were finely ground and powdered. A portion equivalent to 100 μg mL-1 solution was accurately weighed and transferred to volumetric flask and dissolved. Total volume made up to the mark diluting with ethanol. The solution was sonicated for 15 min and centrifuged at 3000 rpm for 10 min, and filtered through a 0.22 μm PTFE syringe filter with Whatman filter paper. An aliquot portion was transferred to volumetric flask, diluted with ethanol as to provide a stock solution of 100 μg mL-1. All stock solutions were stored at 4OC in refrigerator. Dilution has been made to accurately measured aliquots of the stock solution with ethanol to give working concentrations of the analyte.

2.3.2.Blood samples 0.5 mL upper layer of the whole blood after centrifugation were taken in each of three vials. 0.5 mL acetonitrile were added into each vial. For blank solution 1 mL ethanol were added into a vial and remaining two vials were spiked with the addition of 1 mL of 1 and 3 μg mL-1 standard solution. The solution was sonicated for 15 min and centrifuged at 3000 rpm for 10 min, and filtered through a 0.22 μm PTFE syringe filter with Whatman filter paper. All solutions were stored at 4OC in refrigerator before analysis.

2.3.3.Urine Samples

1 mL of urine were taken in each of three vials. For blank solution 1 mL ethanol were added into a vial and remaining two vials were spiked with the addition of 1 mL of 1 and 3 μg mL-1 standard solution. The solution was sonicated for 15 min and centrifuged at 3000 rpm for 10 min, and filtered through a 0.22 μm PTFE syringe filter with Whatman filter paper. All solutions were stored at 4OC in refrigerator before analysis.

2.4. Preparation of Calibration Curve

Calibration curves were construced for five concentration levels of the analyte ranging from 1, 3, 5, 7 and 10 μg μL-1. Peak area of each chromatogram at different levels was plotted against theoretical concentrations. Calibration curve constructed was fitted by a least squares linear regression to the equa- tion, y = mx + c. where, y = response ratio, m = slope, x = concentration, c = intercept. With reference to this calibration equation unknown concentration of the analyte was determined.

2.5. Chromatographic Conditions

A standard solution of 5 μg mL-1 drug was used for the optimization of the chromatographic conditions. All through the experiment a reversed-phase Gemini 3U, C18, 110R (150 × 4.6 mm, 3 μm) column and NaH2 PO4 (dihydrogen sodium phosphate, 5 mM) were used as buffer solution. Special attention have been paid on optimization of the mobile phase composition to gain good resolution avoding tailing of the peak. To detect the absorption maxima a UV scan of standard solution prepared by mobile phase was done in the range of 200 to 400 nm for the spectra of studied drugs (Figure 3). An efficient UPLC method was evaluated by the satisfactory results with good resolution at reduced elution time and tailing problems under this optimized com- position. With respect to sharpness and symmetry of the peaks best flow rate was investigated. Different composition of mobile phase consisting CH3OH, CH3CN and NaH2PO4 under isocratic program was checked as the optimized conditions at a flow rate of 1 mL/ min at ambient temperature. The injection volume was 10 μL. Prior to the analysis buff- er solution was filtered in vacuum using 0.2 μm membrane, mobile phase was degassed by a stream of helium and column was equili brated with the mobile phase.

2.6. Validation Parameters

ICH guidelines were followed for the validation of the method14. In this regard ana- lytical performance parameters precision, ac- curacy, specificity, limit of detection, limit of quantitation, linearity and range, suitability and robustness were studied.

Result and Discussion

3.1. Chromatography

The chromatographic conditions optimized were composition of the solvents, and mobile phase flow rate. Mobile phase it must elute all the different substances with satisfactory peak shape and in a short time. Initial experiments with the LC system using methanol or acetonitrile as organic modifier in the buffered mobile phase were performed for better separation of analytes. The combination of methanol with 5 mM dihydrogen sodium phosphate served our intentions best. Reversed-phase Gemini 3U, C18, 110R (150 × 4.6 mm, 3 μm) column and 5 mM NaH2PO4 as buffer solution all through the experiment were used to study the simultaneous separation of both drugs.

In particular, peak tailing observed was considerable. In order to deter- mine the detection wavelength, the absorption spectra of all compounds were obtained. The absorption spectra of all compound showed absorption bands in the UV region with maximum absorption wave lengths be- tween 235 to 245 nm as shown in Figure 3. Therefore, 240 nm selected for monitoring as compromised to the both drug. To determine the optimum mobile phase flow-rate under optimized composition the effect on Rt, peak height and peak width was studied. As expected when the mobile- phase flow-rate was increased Rt decreased. A flowrate of 1 mL min-1 was chosen as a compromise analysis time, because this value also maintains good peak shape. The mobile phase mixture of CH3OH, CH3CN and 5 mM NaH2PO4 by the composition of 90:05:05 (v/v) was optimized at isocratic program (Table 1). The method was carried out for the detection and quantitation of the drug representing total elution time less than 2.2 min (2.191±0.005 minutes) (Figure 4).

The method developed herein was ap- plied to various concentrations taken from the pharmaceutical products and plasma and urine samples for determining the content of investigated drugs.

Table 2 summarizes intraday and interday precision and accuracy data, indicating that these values are acceptable and the method is accurate and precise. Table 3 shows the validation performance of the proposed UPLC method. Analytical data of system suitability and robustness are placed in Table 4. Table 5 also shows the column efficiency data as the validation evidences.

3.2. Validation parameters


The calibration curves constructed for standard using working concentration at levels 1, 3, 5, 7, 10 μg mL-1 of each drug. Calibration curves were constructed using peak area of drug versus nominal concentrations of the analytes. Calibration equations were y=23172x+2941.4 for KTR and y=14493x+3092.8 for TMS. The calibra-


tion curves were linear in the range of 1-10 μg mL-1 for both KTR and TMS. The coeffi cients of corelation (r2 ) were 0.9997 for both drugs. Figure 5 shows the calibration curves for the determination of KTR and TMS, respectively.


The limit of detection were calculated from calibration graph by the formula; LOD=3•Sxy/a, and the limit of quantifica- tion; LOQ=10•Sxy/a. The LOD and LOQ were found to be 0.125 and 0.41 μg mL-1 for KTR and 0.150 and 0.50 μg mL-1 for TMS respectively. These results indicate that method was sensitive enough for therapeutic assay. 3.2.3.Recovery/Accuracy

The results of recovery obtained from the within-day assay at five concentrations (n=5) by the proposed method was 99.18- 103.34% for KTR and 99.14-101.76% for TMS while between-day assay at six different days was 99.39-102.04% for KTR and 98.08- 103.78% for TMS. The recovery showed that high accuracy of the drug determination. Intraday and interday recovery data of pro- posed method are presented in Table 2.


The relative standard deviations (RSD)

obtained for the within-day assay at five concentrations (n=5) was 0.32-0.99% for KTR and 0.40-1.01% for TMS while betweenday assay was in the range 0.51-2.77% for KTR and 0.41-2.26% for TMS. The precision results showed that the high precision of the method. Intraday and inter-day precision data for proposed method are presented in Table 2. Validation performances of the proposed UPLC method are presented in Table 3.


The specificity showed that drugs were free of interference from potential impurities and degradation products by the absence of any peak in the same retention times. Peak purity of KTR and TMS was passed in standard. From the chromatogram shown in Figure 4, it is evident that under the chosen chromatographic conditions KTR (Tr=2.187 min), TMS (Tr=1.148 min). Results indicate the high specificity of the method and can be used in a stability assay and routine analysis of the investigated drugs.


It was found that the percent recoveries were excellent under most conditions, and remained unaffected by small deliberate changes of experimental parameters including the flow rate and isocratic program (Table 1) though retention time and resolution was shortened as expected. There was no notice- able difference between the chromatograms when the wavelength was varied by ±3 nm. Variation in the experimental parameters (flow rate, isocratic program) provided an in- dication of its reliability during normal use and concluded that the method was robustas shown in Table 4.

3.2.7.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 KTR and TMS to be performed. The system suitability was assessed by replicate injec- tions (n=5) of the sample at 5 μg mL-1 includ- ing within- and between-day assessments for standard. Precision of retention time and peak area was examined to evaluate the system suitability. RSD of the peak area 0.49% for KTR, 0.56% for TMS and that of retention time 0.17 min for KTR and 0.37 min for TMS indicates excellent suitability of the system as shown in Table 4.

3.2.8.Column Efficiency

The column efficiency parameters have been calculated for a representative chromatogram. This test is essential for the assurance of the quality performance of a chromatographic system. The calculated values of theoretical plate number, tailing factor, separation factor, resolution factor and capacity factor as shown in Table 5 revealed the excellent performance of analytical column.

3.3. Applications

3.3.1.Pharmaceutical formulations

The method developed here was ap- plied to various concentrations (3.0, 5.0 μg mL-1) of solutions prepared from pharma- ceutical products for determining the content of KTR and TMS. The values of the overall drug percentage recoveries and the RSD values of measurements are as presented in Table 6 and 7. Determination was free of interference from degradation products and no interference from the sample excipients could be observed at this detection wavelength, indicating the high specificity of the method. Results indicate that measurements are acceptable with good precision. Recovery was almost same as that of levelled values for four

tested samples. Some contain excessive large amount and some contain lower than labelled values. It is may be due to lack of proper quality management.


The method developed here was applied to various spiked concentration of solutions prepared from biological samples, plasma and urine, taken from one volunteer under regular treatment for determining the content of KTR and TMS. Standard addition method was applied to the analysis of biological samples. Regression equation after standard addition yielded both drugs as shown in Table 8 and Table 9. The values of the overall drug percentage recoveries and the RSD values of measurements are presented. The absence of any endogenous interfering peak observed in the extracts of bio-fluids overlapping with any analyte indicates the high specificity of the method which can be used in therapeutic and routine analyses.

3.3.3.Drug Intraction

Study The drug interaction for the concomitant use of KTR and TMS was studied few. No evidences of degradation or interferences was detected at present conditions with in study range or retention time as shown in Figure 6-8. From the chromatogram it is evident that no drug interactions or online derivatization has been occurred in their combined determination, since there was no peaks on their retention times. Peak purity was passed in standard as the excellent RSD precision of area to retention time and area of 10 measurements of 5 μg mL-1 KTR and

TMS. The average retention time for TMS in single measurement of which was 1.14 ± 0.04 min and RSD was 0.34%. The average re-tention time for KTR in single measurement of which was 2.191 ± 0.0037 min and RSD was 0.17%. RSD of area of ten measurements was 0.49% and 0.56% for KTR and TMS, respectively. In mixture the RSD of average retention time and peak area were almost same as that of individual values. Our present study shows that both drugs have been determined simultaneously in their mixture free of inter-

ference from potential impurities and degradation products by the absence of any peak in the same retention times.


The proposed work provides a fast, precise, sensitive, accurate, linear, robust, simple and rugged UPLC assay method. For the proposed method both the drugs gave well define peaks. They were well separated. The validation data demonstrate good precision and accuracy, which prove the reliability of the proposed method. The reproducibility, repeatability and accuracy of the proposed method were found to be satisfactory which is evidenced by low values of standard devia- tion and percent relative standard deviation in comparison to previous methods. No evidences of drug interaction has been detected. Method was successfully validated as per ICH guidelines can be conveniently employed for routine quality control analysis of ketorolac tromithamine and tiemonium methylsulphate in pharmaceutical formulation and biological samples without any interference.

Conflicts of interest

Authors declared no conflicts of interest.



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