We report a selective, accurate, and reproducible liquid chromatography-tandem mass spectrometric (LC-MS/MS) method that employs solid phase extraction for quantification of ketorolac enantiomers in human plasma. Resolution of R(+)-ketorolac and S(−)-ketorolac was achieved using a Chiral-AGP column and a mobile phase of ammonium formate buffer (10 mM, pH
Ketorolac tromethamine is a potent nonnarcotic analgesic compound with cyclooxygenase inhibitory activity which has been developed for oral and parenteral use [
Various methods have been reported for measurement of ketorolac without differentiating between the enantiomers [
The overall aim was to develop a sensitive LC-MS/MS method for simultaneous determination of ketorolac enantiomers in human plasma samples of a bioequivalence study. The present method, as detailed in sections that follow, offers a relatively simple sample preparation procedure using SPE and has been comprehensively validated, offering the advantage of simplicity with adequate sensitivity, selectivity, and precision to determine ketorolac enantiomers in plasma samples. Compared to the LLE method described by Ing-Lorenzini et al. the present method is having advantage of less extraction time and better sample quality due to SPE. Following validation, this assay was successfully applied to a bioequivalence study of ketorolac tromethamine to quantify its enantiomers.
R(+)-ketorolac (98.09% pure), S(−)-ketorolac (99.28% pure), and S(+)-etodolac (internal standard; 99.23% pure) used to prepare stock solutions were purchased from Varda Biotech (P) Ltd., India. Ammonium formate was purchased from Fluka (Buchs, Switzerland). Formic acid (85% pure) and methanol (99.8% pure) was procured from Fischer Scientific (India). HPLC grade acetonitrile from Spectrochem (India) was used for the preparation of mobile phase. Water was procured in-house using a Milli-Q device (Millipore, Moscheim Cedex, France). K3 EDTA (Ethylene diamine tetra acetic acid) containing plasma batches was obtained from Yash Laboratories, Pune, India. Figure
Structures of R(+)-ketorolac, S(−)-ketorolac, and S(+)-Etodolac.
The instrumentation consisted of a modular HPLC (Shimadzu, Kyoto, Japan) coupled to AB Sciex API-3200 mass spectrometer (Applied Biosystems, Ontario, Canada), equipped with an electrospray ion interface. The HPLC system consisted of two LC-20AD pumps (identified as pumps A and B), a CTO-20A column oven, a SIL-HTc autosampler, 20A semi-micromixer having mixing volume 100
During method development, chromatographic resolution was optimized on a Chiral-AGP (
Gradient Time Program.
Time (min) | Events | Flow (ml/min) | |
% Mobile phase A | % Mobile phase B | ||
0.01 | 100 | 0 | 0.5 |
8.00 | 100 | 0 | 0.5 |
9.00 | 0 | 100 | 0.5 |
9.50 | 0 | 100 | 0.8 |
13.50 | 0 | 100 | 0.8 |
14.00 | 100 | 0 | 0.8 |
19.80 | 100 | 0 | 0.8 |
19.90 | 100 | 0 | 0.5 |
20.00 | STOP |
The mass spectrometer was operated in positive turbo-ion spray mode. Multiple Reaction Monitoring (MRM) mode was used to monitor
Optimized ion source and compound parameters.
Parameter | R(+)-Ketorolac | S(−)-Ketorolac | S(+)-Etodolac |
---|---|---|---|
Declustering potential | 41.00 V | 41.00 V | 32.00 V |
Entrance potential | 10.00 V | 10.00 V | 10.00 V |
Collision energy | 25.00 V | 25.00 V | 19.00 V |
Clustering cell exit potential | 2.00 V | 2.00 V | 2.00 V |
Temperature | 550 | 550 | 550 |
Collision associated dissociation | 5 | 5 | 5 |
Curtain gas | 25 | 25 | 25 |
Ion spray voltage | 1500 | 1500 | 1500 |
Chromatograms were acquired using Analyst software (version 1.4.1, Applied Biosystems, Ontario, Canada). A calibration curve is constructed using peak area ratios (PARs) of the calibration standards by applying linear, 1/concentration squared weighted, least squares regression algorithm. All concentrations are then calculated from their PARs against the calibration line.
Separate stock solutions of R(+)-ketorolac, S(−)-ketorolac, and S(+)-etodolac were prepared by dissolving accurately weighed standards in methanol to yield the concentrations of 989021.85 ng/ml,1103318.50 ng/ml, and 110292.61 ng/ml, respectively, and stored in polypropylene container. The concentrations were corrected for purity, moisture content and amount weighed as per certificates of analysis.
Working solutions for calibration curve (CC) standards and quality control samples (QC) were prepared in methanol: water (50 : 50, v/v). The stock solutions of R(+)-ketorolac and S(−)-ketorolac were used to prepare working solutions. These dilutions were spiked in K3 EDTA plasma. Each calibration curve consisted of one blank sample, one blank sample fortified with IS, and eight calibration points ranging from 9.36 to 1198.69 ng/ml for R(+)-ketorolac and from 6.07 to 776.74 ng/ml for S(−)-ketorolac. The QC samples spiked independent of CC standards comprised Lower Limit of Quantification (LLOQ), Low-Quality Control (LQC), Middle-Quality Control (MQC), and High-Quality Control (HQC). Aliquots of the CC and QC were stored below −50°C. Figure
Representative calibration curves R(+)-ketorolac and S(−)-ketorolac.
Plasma samples frozen at −50°C were thawed on the day of extraction at room temperature followed by vortexing to ensure homogeneity. IS dilution (50
All validation exercises were conducted in conformance to in-house standard operating procedures and the U.S. FDA Guidance document [
Result of validation parameters.
Validation parameter | R(+)-Ketorolac | S(−)-Ketorolac |
---|---|---|
Internal standard | S(+)-Etodolac | S(+)-Etodolac |
Bench top stability (in plasma samples) | 9.02 hr at room temperature | 9.02 hr at room temperature |
Autosampler stability | 110.67 hr at | 110.67 hr at |
Stock solution stability of the drug stored at refrigerated temperature between 1–10 | 11 days | 11 days |
Carry over effect in matrix | 0% at RT of analyte and IS | 0% at RT of analyte and IS |
Stock stress stability in aqueous dilutions | 8.95 hr at room temperature under low light condition in poly propylene container. | 8.95 hr at room temperature under low light condition in poly propylene container. |
Recovery | 82.04% | 70.94% |
Freeze thaw stability | 3 cycle | 3 cycle |
Dilution integrity | Dilution at 2 times and 4 times | Dilution at 2 times and 4 times |
Limit of quantitation (ng/ml) | 9.36 | 6.07 |
Analytical range (ng/ml) | 9.36–1198.69 | 6.07–776.74 |
Long-term stability-1 | 117 days at temperature below | 117 days at temperature below |
Long-term stability-2 | 283 days at temperature below | 283 days at temperature below |
The objective of this study was to chromatographically resolve enantiomers of ketorolac to enable accurate quantification. Two chiral analytical columns, namely, Chirobiotic V2 (
Baseline chromatographic resolution could not be achieved on the Chirobiotic V2 column with a mobile phase ammonium formate buffer (10 mM, pH 4.00–5.50): acetonitrile (70 : 30, v/v). However, a good resolution was observed on the Chiral AGP column using ammonium formate buffer (10 mM, pH 4.70): acetonitrile.
In order to undertake successful quantification of enantiomers, tuning parameters for ESI+ were optimized for the protonated precursor and product ions of analytes and IS. R(+)-ketorolac, S(−)-ketorolac, and S(+)-etodolac (IS) were found to have retention time of
(a), (b), and (c) represent chromatograms of drug-free plasma, drug-free plasma fortified with internal standard, and limit of quantification samples, respectively.
Linearity of R(+)-ketorolac and S(−)-ketorolac determination was established over a concentration range of 9.36–1198.69 ng/ml and 6.07–776.74 ng/ml, respectively, in spiked human plasma. The selected standard calibration range covered the therapeutic levels of drug in human plasma samples. Linear coefficient of regression (
Three precision and accuracy batches were run to check intra- and interday precision and accuracy. The results for precision and accuracy are summarized in Table
Intra- and interday precision and accuracy for determination of ketorolac enantiomers in human plasma by LC-MS/MS.
Nominal concentration (ng/ml) | Observed ( | %CV | %Accuracy | Number of observations ( |
---|---|---|---|---|
Intra-day | ||||
9.37 (LOQQC) | 3.5 | 100.7 | 12 | |
24.57 (LQC) | 3.2 | 97.7 | 12 | |
511.90 (MQC) | 3.0 | 102.6 | 12 | |
984.42 (HQC) | 2.4 | 98.8 | 12 | |
6.09 (LOQQC) | 4.1 | 97.7 | 12 | |
15.95 (LQC) | 3.9 | 89.3 | 12 | |
332.35 (MQC) | 2.4 | 94.5 | 12 | |
639.14 (HQC) | 1.7 | 89.6 | 12 | |
Inter-day | ||||
9.37 (LOQQC) | 6.3 | 99.5 | 18 | |
24.57 (LQC) | 4.1 | 98.4 | 18 | |
511.90 (MQC) | 5.2 | 96.9 | 18 | |
984.42 (HQC) | 4.7 | 98.9 | 18 | |
6.09 (LOQQC) | 4.7 | 97.0 | 18 | |
15.95 (LQC) | 6.7 | 91.5 | 18 | |
332.35 (MQC) | 5.2 | 96.9 | 18 | |
639.14 (HQC) | 5.5 | 92.1 | 18 |
The recovery/ for R(+)-ketorolac, S(−)-ketorolac, and S(+)-etodolac was calculated by comparing the peak areas of processed plasma which was prespiked with analytes at low, medium, and high concentration levels, with peak area of aqueous mixture of analytes representing 100% extraction of samples at low, medium, and high concentration levels. The mean extraction recovery of R(+)-ketorolac, S(−)-ketorolac, and S(+)-etodolac was 82.04, 70.94, and 93.9%, respectively.
Selectivity was performed using nine different lots of K3-EDTA plasma. From each lot, a single aliquot was processed along with six aliquots of the lower limit of quantification sample spiked in K3-EDTA plasma. There was no significant interference observed at the retention times of analytes and internal standard.
The samples for dilution integrity were spiked at a concentration approximately two times the concentration of 90% ULOQ (upper limit of quantification). An appropriate volume of this sample was diluted 2-fold and 4-fold using blank matrix. Six replicates at each dilution level were analyzed. The % Accuracy was found to be 104.1 for R (+)-ketorolac and 113.6 % for S(−) Ketorolac. The Precision was found to be 3.0 for both R(+)-ketorolac and S(−) Ketorolac, respectively.
In order to determine the matrix effect, nine lots of drug free plasma were chosen and concentrations equivalent to LQC and HQC levels were spiked in each lot. At each level, samples were processed in duplicate. The value of QC samples was back calculated against the freshly spiked calibration curve. CV of 3.1%–3.3% and 3.4%–3.5% and accuracy between 95.3%–101.9% and 92.7%–99.4% were observed for R(+)-ketorolac and S(−)-ketorolac, respectively.
For the calculation of matrix factor, working solutions of drug and IS were prepared at concentrations representing 100% extraction of QC samples at low, middle, and high concentrations (aqueous samples). Four replicates each of working solutions of drug and IS at Low, Middle, and High QC levels were run from the same respective vial at each QC level (Total 12 samples). Thereafter, twelve aliquots of screened matrix lots were taken and processed. Every four aliquots were reconstituted with working solutions of drug and IS at Low, Middle and High QC levels respectively. Matrix factor (MF) was calculated at low, middle, and high QC levels by comparing the mean peak area ratio of matrix samples reconstituted with aqueous dilution to that of mean peak area ratio of aqueous samples. The matrix factor of R(+)-ketorolac ranged from 0.97 to 1.01 and for S(−)-ketorolac ranged from 0.96 to 1.00. These values are indicative of neither any ion-suppression nor ion-enhancement. The CV of matrix factor between low middle and high QC levels was found to be 2.5 and 2.7% for R(+)-ketorolac and S(−)-ketorolac, respectively.
Stock solution stability of R(+)-ketorolac, S(−)-ketorolac, and S(+)-etodolac was determined for 11 days. The stocks were stored in a refrigerator between 1 and 10
The results of freeze thaw stability, bench top stability, autosampler stability, and long-term stability are summarized in Table
Freeze thaw stability, bench top stability, autosampler stability, and long-term stability data of R(+)-ket orolac and S(−)-ketorolac in human Plasma.
Storage period and condition | Analyte | Nominal | CV(%) | %Accuracy | %Stability | ||
---|---|---|---|---|---|---|---|
Sample type | Conc.(ng/mL) | Mean | |||||
Freeze thaw cycles(3 cycles) | R(+)-ketorolac | LQC | 24.57 | 24.46 | 3.2 | 99.5 | 101.1 |
HQC | 984.42 | 1015.94 | 3.2 | 103.2 | 102.5 | ||
S(−)-ketorolac | LQC | 15.95 | 15.2 | 3.9 | 95.3 | 99.6 | |
HQC | 639.14 | 625.65 | 4.4 | 97.9 | 101.6 | ||
Bench top stability in plasma samples (9.02 hours) | R(+)-ketorolac | LQC | 24.57 | 26.57 | 3.6 | 108.2 | 104.5 |
HQC | 984.42 | 959.49 | 2.5 | 97.5 | 101 | ||
S(−)-ketorolac | LQC | 15.95 | 15.77 | 4.2 | 98.8 | 103.7 | |
HQC | 639.14 | 587.9 | 2.4 | 92 | 102.8 | ||
Autosampler stability (110.67 hours) | R(+)-ketorolac | LQC | 24.57 | 23.15 | 3.7 | 94.2 | 97.5 |
HQC | 984.42 | 949.18 | 1.2 | 96.4 | 99 | ||
S(−)-ketorolac | LQC | 15.95 | 14.19 | 3.9 | 88.9 | 97 | |
HQC | 639.14 | 571.73 | 1.8 | 89.5 | 97.2 | ||
Long term stability 1 (117 Days) | R(+)-ketorolac | LQC | 24.57 | 26.84 | 2.9 | 109.2 | 113 |
HQC | 984.42 | 1034.84 | 1.2 | 105.1 | 99.5 | ||
S(−)-ketorolac | LQC | 15.95 | 17.39 | 3.7 | 109 | 109.9 | |
HQC | 639.14 | 665.13 | 1.1 | 104.1 | 99.1 | ||
Long term stability 2 (283 Days) | R(+)-ketorolac | LQC | 24.57 | 25.72 | 1.2 | 104.7 | 101.3 |
HQC | 984.42 | 977.32 | 2.4 | 99.3 | 99 | ||
S(−)-ketorolac | LQC | 15.95 | 17.39 | 1.1 | 104.5 | 101.2 | |
HQC | 639.14 | 665.13 | 2.8 | 98.6 | 100 |
Appropriate dilutions of analytes, internal standard, and reference dilution were prepared from the respective standard stock solutions and stored for 8.95 hours at room temperature in polypropylene container. After this stability period, fresh dilutions of the analytes, internal standard, and reference dilution were prepared (comparison dilutions) from the same respective standard stock solutions from which the stability dilutions were made. Six replicates each of stability dilutions and comparison dilutions were injected from the same vial. The dilutions were found to be stable for the storage period evaluated with percent stability (%CV) of 103.0 (0.7) and 100.2 (0.6) for R(+)-ketorolac and S(−)-ketorolac, respectively.
Two blank plasma samples, two LLOQ samples, and two ULOQ plasma samples were processed and injected in the following order. One Blank sample was injected, followed by two LLOQ samples, two ULOQ samples, and one blank sample. Percentage carry-over was calculated with respect to the mean of two LLOQ areas at RT of analyte and IS.
It was found that there is no carry-over at the RT of analytes and IS.
This method was successfully applied to an open label, balanced, randomized, two-period, two-sequence, single dose, crossover, bioequivalence study of ketorolac involving twenty-two healthy male volunteers following oral administration of 10 mg of ketorolac tromethamine mouth dissolving tablet of Ranbaxy Laboratories Limited (test) under fasting condition and the same was compared with the TORADOL 10 mg tablet (containing 10 mg ketorolac tromethamine) of Roche Products Limited (reference).
All healthy Indian human volunteers were in the age range of 18–45 years, medically examined, and had voluntarily provided their written informed consent before initiation of study. The study protocol and written informed consent were approved by the ethical committee of Jamia Hamdard Institutional Review Board, New Delhi, India.
Venous blood samples were collected in K3 EDTA tubes predose and at 0.083, 0.167, 0.250, 0.333, 0.500, 0.667, 0.833, 1.000, 1.500, 2.000, 2.500, 3.000, 4.000, 6.000, 8.000, 12.000, 16.000, 20.000, and 24.000 h after dosing. Plasma was separated by centrifugation and the separated plasma samples were stored below −50°C until analysis. Following analysis, pharmacokinetic parameters were calculated by noncompartmental analysis using WinNonlin Professional software (Version 5.0, Pharsight Corp., Mountain View, CA, USA). The peak plasma concentration (
Pharmacokinetic parameters (mean ± SD) of ketorolac tromethamine mouth dissolving tablets (10 mg) based on its plasma concentrations.
Parameters ( | Tmax (h) | Cmax (ng/ml) | AUC0→t (h ng/ml) | AUC0→∞ (h ng/ml) | ||||
R | T | R | T | R | T | R | T | |
R(+)-Ketorolac | ||||||||
S(−)-Ketorolac |
R: Reference product; T: Test product; values represented as
Linear plot of mean plasma R(+)-ketorolac concentration (ng/ml) versus time (h) in healthy adult human male subjects (
Linear plot of mean plasma S(−)-ketorolac concentration (ng/ml) versus time (h) in healthy adult human male subjects (
The mean Cmax of R(+)-ketorolac was
An Incurred Sample Reanalysis (ISR) was performed on 90 sample points from 15 different subjects selected randomly using statistical analysis software (SAS). Three time points from each period of these identified 15 subjects were selected for ISR. One sample was at the
Confirmatory incurred sample reanalysis of R(+)-ketorolac and S(−)-ketorolac.
R(+)-Ketorolac | S(−)-Ketorolac | |
---|---|---|
No. of total samples taken for ISR | 90 | 90 |
No. of samples meeting the acceptance criteria (i.e., % difference between original and reanalyzed value must be within 20%) | 82 | 76 |
% of samples meeting the acceptance criteria | 91.1% | 84.4% |
Bland-Altman plot of the data obtained from ISR of R(+)-ketorolac.
Bland-Altman plot of the data obtained from ISR of S(−)-ketorolac.
A simple and accurate chiral LC-MS/MS method was described for the enantiomeric separation of ketorolac. Chiral AGP, a