TY - JOUR
AU - Hasan, Mohamed, A
AB - Abstract A sensitive, selective, accurate and precise ultra-high performance liquid chromatography–Tandem mass spectrometry (MS/MS) method was developed and validated for the simultaneous determination of drugs used as eye drops in cataract surgery in aqueous humor. Cataract surgery requires a powerful mydriatic eye drops combination such as cyclopentolate hydrochloride and phenylephrine hydrochloride to dilate the pupil and facilitate eye lens replacement and also requires strong fluoroquinolone antibiotic such as lomefloxacin hydrochloride. The method was performed with positive ion electrospray ionization and the analytes were quantified and monitored on a triple quadrupole mass spectrometer using multiple reaction monitoring scanning mode. Liquid–liquid extraction was used for the purification and preconcentration of analytes from rabbit aqueous humor matrix. Chromatographic elution was performed using an Phenomenex Luna® C18 (150 mm × 2.1 mm, 1.6 μm) column and moxifloxacin hydrochloride as internal standard with a mobile phase consisting of methanol:water:formic acid (70:29:1, by volume) at flow rate of 0.2 mL/min. Satisfactory results regarding linearity, recovery, stability, accuracy and precision of the analytes were obtained. Full validation of the procedure was performed according to the US Food and Drug Administration guidance for industry: bioanalytical method validation and European Medicines Agency (EMA) guideline on bioanalytical method validation. Introduction Cataract causes about 51% of blindness and 33% of cases of vision impairment in the world. Cataract surgery is the most commonly used and effective treatment, which includes the removal of the natural lens of the eye that has developed an opacification then an artificial intraocular lens implant is inserted (1). Cataract surgery requires a powerful mydriatic agents combination such as cyclopentolate hydrochloride, which is an antimuscarinic drug (Figure 1A) and phenylephrine hydrochloride, which is selective α1-adrenergic receptor agonist (Figure 1B) eye drops to dilate the pupil and facilitate eye lens replacement (2). The surgery also requires a strong antibiotic such as lomefloxacin hydrochloride (Figure 1C) eye drops, which is fluoroquinolone antibacterial agent. Lomefloxacin hydrochloride enters bacteria through porin channels and exhibit antimicrobial effects on DNA gyrase (bacterial topoisomerase II) and bacterial topoisomerase IV with a wide spectrum of activity (2). The aqueous humor is a transparent, watery fluid similar to plasma, but containing low protein concentration. It is secreted from the ciliary epithelium, a structure supporting the lens (3). It fills both the anterior and the posterior chambers of the eye. Aqueous humor is composed of amino acids, electrolytes, ascorbic acid, lactic acid, glutathione, immunoglobulins and 98% water (4). Figure 1 Open in new tabDownload slide Chemical structures and chemical names of cyclopentolate hydrochloride (A), phenylephrine hydrochloride (B), lomefloxacin hydrochloride (C), moxifloxacin hydrochloride (D). Figure 1 Open in new tabDownload slide Chemical structures and chemical names of cyclopentolate hydrochloride (A), phenylephrine hydrochloride (B), lomefloxacin hydrochloride (C), moxifloxacin hydrochloride (D). Literature surveys revealed that lomefloxacin hydrochloride has been determined in biological fluids including aqueous humor, plasma, urine and milk (5–9). Cyclopentolate hydrochloride has been determined in eye drops and pharmaceutical preparations (10–12), whereas phenylephrine hydrochloride has been determined in aqueous humor, eye drops and pharmaceutical preparations (13–16). Most of the previously mentioned methods are not sensitive enough to be used for the determination of the cited drugs in aqueous humor, especially due to the complex nature of the biological matrices as aqueous humor matrix and the low concentrations of the three drugs in the aqueous humor samples, which require the development of sensitive and selective method such as Liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Application of LC–MS/MS deemed useful for the analysis of the cited drugs due to better selectivity, sensitivity, peak assignment, structural information, chromatographic integrity and rapid method development, sample preparation and analysis with a smaller sample size compared with conventional high-performance liquid chromatography (HPLC) coupled with ultraviolet detection that permits simultaneous determination of several analytes. Therefore, the main objective of this part was to develop LC–MS/MS method for the analysis of certain drugs used as eye drops in cataract surgery namely; cyclopentolate hydrochloride, phenylephrine hydrochloride and lomefloxacin hydrochloride in spiked rabbit aqueous humor using moxifloxacin hydrochloride as internal standard (IS) (Figure 1D). Experimental Materials Pure cyclopentolate hydrochloride (99.45%) was kindly supplied by Kahira Pharmaceutical and Chemical Industrial Company, Cairo, Egypt. Pure phenylephrine hydrochloride (99.70%) was kindly supplied by Kahira Pharmaceutical and Chemical Industrial Company, Cairo, Egypt. Pure lomefloxacin hydrochloride (99.65%) was kindly provided by Sigma Pharmaceutical Industries, Quesna City, Egypt, S.A.E. Pure moxifloxacin hydrochloride as IS (99.45%) was kindly supplied by EVA Pharmaceutical Industrial Company, Cairo, Egypt. Chemicals and reagents Acetonitrile, ammonium acetate, dichloromethane, diethyl ether, ethyl acetate, formic acid and methanol, HPLC grade (Sigma-Aldrich, Germany). Water used throughout the procedure was freshly distilled. Apparatus Ultra-high performance liquid chromatography (UPLC) MS/MS “Waters” 3100 USA equipped with Phenomenex Luna® Omega C18 column (150 mm × 2.1 mm, 1.6 μm), TQ detector (Acquity ultra-performance LC), binary solvent manager pump (Acquity ultra-performance LC) and auto sampler (sample manager Acquity ultra-performance LC). The chromatographic analysis was carried out using (Mass lynx V4.1.) data analysis program. Benchtop centrifuge (TDL-60B) with maximum speed 6000 rpm (Hunan, China, Mainland). Rotary evaporator (Scilogex-RE 100-pro, USA). Analytical balance (Precisa125A, Switzerland). Standard solutions Standard stock solutions (100 μg/mL) of each drug were prepared separately in methanol by dissolving an accurately weighed amount of the drug and adding 50 mL of methanol, then the mixture was sonicated and completed to volume in a 100-mL volumetric flask. Working solutions of each drug were prepared at 0.01, 0.5, 1, 5, 7.5 and 10 μg/mL by serial dilution of the standard stock solution of each drug with methanol. The IS stock solution was prepared in methanol at concentration of 100 μg/mL. And the working solution of IS was prepared at (10 μg/mL) by diluting the standard stock solution of IS with methanol. All solutions were stored under refrigeration (2–8°C) and in the dark for optimum stability when not in use. Procedures Chromatographic and tandem mass conditions Chromatographic elution was performed using a Phenomenex Luna® C18 (150 mm × 2.1 mm, 1.6 μm) column with a mobile phase consisting of methanol:water:formic acid (70:29:1, by volume) pumped through the column at flow rate of 0.2 mL/min. Under these conditions, the total run time was 7 minutes and the injection volume was 10 μL. Method parameters including flow-dependent parameters and compound-dependent parameters were optimized. Ion spray voltage was 3 kV and ion spray temperature was kept at 200°C. Nebulizer gas (Gas 1) and heater gas (Gas 2) both were set at 50 psi. Collision energies were 15 eV for cyclopentolate hydrochloride and phenylephrine hydrochloride and 20 eV for lomefloxacin hydrochloride and moxifloxacin hydrochloride (IS). Cone volt of 20 V for cyclopentolate hydrochloride and phenylephrine hydrochloride and 50 V for lomefloxacin hydrochloride and moxifloxacin hydrochloride (IS). The analysis was carried out utilizing multiple reaction monitoring in the positive ion mode for all the chosen drugs and the IS at m/z 292.19 → 274.18 for cyclopentolate hydrochloride, m/z 168.10 → 150.09 for phenylephrine hydrochloride, m/z 352.15 → 308.16 for lomefloxacin hydrochloride and m/z 402.18 → 384.17 for moxifloxacin hydrochloride (IS), respectively. Aqueous humor collection New Zealand albino rabbits of both sexes (weighing 2–2.3 kg) free of any signs of ocular inflammation or any clinical observable abnormalities were used. In order to collect the aqueous humor, the rabbits were systemically anesthetized with I.M. injections of ketamine hydrochloride (35 mg/kg) in combination with a muscle-relaxing agent xylazine hydrochloride (5 mg/kg). Aqueous humor samples were withdrawn by anterior chamber paracentesis using an insulin syringe (1 mL). The aqueous humor samples were stored in a freezer at −20°C until analysis. General procedure Ten μL of working solutions of each drug and IS were mixed together, then 1.5 mL of diethyl ether were added to the mixture, vortexed for 1 minute and centrifuged at 4000 rpm for 10 minutes. The organic layer (1 mL) was accurately transferred to another tube, evaporated, then reconstituted by adding 100 μL of mobile phase and transferred to a glass vial for LC–MS/MS analysis following the above-mentioned specific conditions. Quality control (spiked) samples preparation The quality control samples (QC) were prepared similarly at three concentration levels: low, medium and high (50, 500 and 750 ng/mL) using the general procedure of the method after addition of 100 μL of rabbit aqueous humor to the mixture for liquid–liquid extraction. Validation of the procedure Full validation of the procedure was performed according to the current US Food and Drug Administration guidance for industry: bioanalytical method validation (17) and EMA guideline on bioanalytical method validation (18). Linearity (construction of the calibration graphs) The general procedure of the method was repeated. Calibration graph was constructed by plotting the peak area ratio (peak area of cited drug/peak area of IS) against the corresponding concentration of each drug in ng/mL. Limits of detection and quantitation The limit of detection (LOD) and the limit of quantitation (LOQ) were calculated based on the signal-to-noise (S/N) ratio, which was performed by comparing measured signals from samples with known low concentrations of analyte with those of blank samples and establishing the minimum concentration at which the analyte can be reliably detected. S/N ratio of 3:1 is generally considered acceptable for LOD. S/N ratio of 10:1 is generally considered acceptable for LOQ. Accuracy and precision (interday and intraday) The accuracy and precision (repeatability) were evaluated by six replicate analysis of drugs mixture at four concentration levels covering the linearity range of the drugs (1, 50, 500 and 750 ng/mL) in the same day. The accuracy and intermediate precision were assessed by six replicate analysis of drugs mixture at four concentration levels covering the linearity range of the drugs (1, 50, 500 and 750 ng/mL) on three consecutive days. The precision of the method was presented as the percent coefficient of variation (%CV). Selectivity Six randomly selected drug-free rabbit aqueous humor samples collected from different animals were processed by the similar liquid–liquid extraction procedure and analyzed as blank samples to determine the extent to which endogenous rabbit aqueous humor components may contribute to the interference at retention time of the analytes and the IS. Extraction recovery and matrix effect Comparative analysis on three sets of samples was based on approach proposed by Matuszewski et al. (19). In the first set (A), three concentrations level (at low quality control [LQC], medium quality control [MQC] and high quality control [HQC] levels) for the neat standards were prepared in the mobile phase and were analyzed in six replicates by the proposed LC–MS/MS. In the second set (B), samples of blank rabbit aqueous humor that were free of any significant interference at the retention time of the standards were extracted by the proposed liquid–liquid extraction procedure and then spiked with the three levels of standards. In the third set (C), the standards at the same three concentration levels were treated in the same manner as for QCs (i.e., standards were spiked to blank rabbit aqueous humor before the extraction procedure). Stability of analytes Stability studies of the selected drugs in the rabbit aqueous humor matrix were fully investigated using LQC, MQC and HQC samples that were analyzed immediately after preparation and after the application of the storage conditions that are to be evaluated. The QC samples were analyzed against a freshly prepared calibration curve of each analyte and then the obtained concentrations were compared with the nominal ones. Short-term stability: QC samples were kept at ambient temperature (20–25°C) for 6 hours and at the end of the 6 hours the samples were processed, analyzed and compared with nominal concentrations. Long-term stability: QC samples were stored in a deep freezer at −20°C for 35 days. At the end of the 35 days, the samples were processed, analyzed and compared with nominal concentrations. Freeze and thaw stability: QC samples were stored at −20°C and subjected to three freeze and thaw cycles. At each cycle, samples were frozen for at least 12 hours before they are thawed unassisted. After the completion of third cycle, the samples were processed, analyzed and results were compared with nominal values. Results Optimization of experimental conditions LC conditions In order to carry out determination of cyclopentolate hydrochloride, phenylephrine hydrochloride and lomefloxacin hydrochloride by isocratic elution using a reversed phase Phenomenex Luna® Omega C18 column as a stationary phase, different mixtures of methanol, ammonium acetate and water in different ratios were tried as a mobile phase but each time we face a problem, such problems include tailing and splitting of the peaks. But by replacing ammonium acetate with formic acid, the previous problems were diminished. So, the optimum ratios of mobile phase used throughout the procedures consists of methanol:water:formic acid (70:29:1, by volume), since it allow the determination of cyclopentolate hydrochloride, phenylephrine hydrochloride and lomefloxacin hydrochloride within a short analytical run time (7 minutes) with shaper peaks and excellent sensitivity. The effect of flow rate on the determination of cyclopentolate hydrochloride, phenylephrine hydrochloride and lomefloxacin hydrochloride was studied. Different flow rates were chosen such as 0.15, 0.2 and 0.25 mL/min and injection volume was 10 μL. A flow rate of 0.2 mL/min was found to be the optimal one that allows the determination within a reasonable time; neither peak broadening, nor splitting were observed. Then, after adjustment of the chromatographic conditions, well-defined symmetrical peaks were achieved, in the total ion chromatograms for cyclopentolate hydrochloride, phenylephrine hydrochloride, lomefloxacin hydrochloride and moxifloxacin hydrochloride (IS) as shown in (Figures 2–5), respectively. From this chromatograms, the peaks of the selected drugs were sharply appeared at retention times of 1.60 ± 0.07, 1.56 ± 0.11, 1.56 ± 0.09 and 1.58 ± 0.12 minutes for cyclopentolate hydrochloride, phenylephrine hydrochloride, lomefloxacin hydrochloride and moxifloxacin hydrochloride (IS) respectively. Figure 2 Open in new tabDownload slide MRM chromatograms of a 10 µL injection of (A) standard cyclopentolate hydrochloride (750 ng/ml) and (B) a blank aqueous humor. Figure 2 Open in new tabDownload slide MRM chromatograms of a 10 µL injection of (A) standard cyclopentolate hydrochloride (750 ng/ml) and (B) a blank aqueous humor. Figure 3 Open in new tabDownload slide MRM chromatograms of a 10 µL injection of (A) standard phenylephrine hydrochloride (750 ng/ml) and (B) a blank aqueous humor. Figure 3 Open in new tabDownload slide MRM chromatograms of a 10 µL injection of (A) standard phenylephrine hydrochloride (750 ng/ml) and (B) a blank aqueous humor. Figure 4 Open in new tabDownload slide Figure 4 Open in new tabDownload slide Figure 5 Open in new tabDownload slide MRM chromatograms of a 10 µL injection of (A) 1000 ng/ml of moxifloxacin hydrochloride (IS) and (B) a blank aqueous humor. Figure 5 Open in new tabDownload slide MRM chromatograms of a 10 µL injection of (A) 1000 ng/ml of moxifloxacin hydrochloride (IS) and (B) a blank aqueous humor. MS/MS conditions The protonated molecule [M + H]+ for each compound was selected as precursor ion. Precursor ions and product ions useful for quantitation and confirmation for cyclopentolate hydrochloride (m/z 292.19 → 274.18), phenylephrine hydrochloride (m/z 168.10 → 150.09), lomefloxacin hydrochloride (m/z 352.15 → 308.16) and for moxifloxacin hydrochloride (m/z 402.18 → 384.17) (IS) as shown in (Figure 6). Figure 6 Open in new tabDownload slide Mass spectra of (A) cyclopentolate hydrochloride, (B) phenylephrine hydrochloride, (C) lomefloxacin hydrochloride and (D) moxifloxacin hydrochloride detected by LC/MS/MS using multiple reaction monitoring (MRM). Figure 6 Open in new tabDownload slide Mass spectra of (A) cyclopentolate hydrochloride, (B) phenylephrine hydrochloride, (C) lomefloxacin hydrochloride and (D) moxifloxacin hydrochloride detected by LC/MS/MS using multiple reaction monitoring (MRM). Preliminary fragmentation experiments were performed to find out the optimal instrumental conditions for their unequivocal identification and confirmation of the QC samples as well as the sensitivity to detect traces level concentration of the analytes. Flow-dependent parameters Different operational conditions for the fragmentation were optimized, the optimum values for nebulizer gas (Gas 1) and heater gas (Gas 2) were 50 psi, for ion spray voltage was 3 kV and ion spray temperature was 200°C. Compounds-dependent parameters The optimum values of collision energies were 15 eV for cyclopentolate hydrochloride and phenylephrine hydrochloride and 20 eV for lomefloxacin hydrochloride and moxifloxacin hydrochloride (IS). The optimum values of cone volt were 20 V for cyclopentolate hydrochloride and phenylephrine hydrochloride and 50 V for lomefloxacin hydrochloride and moxifloxacin hydrochloride (IS). The optimum values of declustering potential, curtain gas and entrance potential were 40 V, 30 psi and 10 V, respectively, for the three analytes and the IS. These conditions ensure maximum sensitivity for quantitation of the selected drugs. Sample extraction optimization Upon trying to get good recovery results for cited drugs, we tried different extraction solvents including diethyl ether, dichloromethane, ethyl acetate and different mixtures of these solvents. Good recovery results were obtained with the use of 1.5 mL of diethyl ether as the extraction solvent and for protein precipitation. No acidification or alkalinization of aqueous humor sample was required for the extraction process with the advantage of achieving better sensitivity, optimum peak shape and faster processing of aqueous humor samples with good recovery results. Method validation As per acceptance criteria, the deviation of each concentration level from the nominal concentration was expected to be within ±15% for both % accuracy and %CV of all of the QC levels of low, medium and high (17, 18). Linearity and range Under the described experimental conditions, the calibration graphs for the three analytes were constructed by plotting the peak area ratio versus drug concentrations in ng/mL. Based on the Cmax values of the cited drugs, a suitable linearity range of 1–1000 ng/mL was selected for them. The regression data were presented in Table I. The values of coefficient of determination indicated the good linearity of the calibration graph. Table I Regression parameters for the determination of cyclopentolate hydrochloride, phenylephrine hydrochloride and lomefloxacin hydrochloride by the proposed LC–MS/MS method Parameters . Cyclopentolate hydrochloride . Phenylephrine hydrochloride . Lomefloxacin hydrochloride . Linearity range (ng/mL) 1–1000 1–1000 1–1000 LOD (ng/mL) 0.293 0.278 0.268 LOQ (ng/mL) 0.971 0.926 0.894 Regression equation y* = bx** + a  Slope (b) 0.0192 0.0241 0.0035  Intercept (a) 0.1658 0.1713 0.0026 Coefficient of determination (r2) 0.9995 0.9995 0.9996 Parameters . Cyclopentolate hydrochloride . Phenylephrine hydrochloride . Lomefloxacin hydrochloride . Linearity range (ng/mL) 1–1000 1–1000 1–1000 LOD (ng/mL) 0.293 0.278 0.268 LOQ (ng/mL) 0.971 0.926 0.894 Regression equation y* = bx** + a  Slope (b) 0.0192 0.0241 0.0035  Intercept (a) 0.1658 0.1713 0.0026 Coefficient of determination (r2) 0.9995 0.9995 0.9996 *Peak area ratio. **Concentration in ng/mL. Open in new tab Table I Regression parameters for the determination of cyclopentolate hydrochloride, phenylephrine hydrochloride and lomefloxacin hydrochloride by the proposed LC–MS/MS method Parameters . Cyclopentolate hydrochloride . Phenylephrine hydrochloride . Lomefloxacin hydrochloride . Linearity range (ng/mL) 1–1000 1–1000 1–1000 LOD (ng/mL) 0.293 0.278 0.268 LOQ (ng/mL) 0.971 0.926 0.894 Regression equation y* = bx** + a  Slope (b) 0.0192 0.0241 0.0035  Intercept (a) 0.1658 0.1713 0.0026 Coefficient of determination (r2) 0.9995 0.9995 0.9996 Parameters . Cyclopentolate hydrochloride . Phenylephrine hydrochloride . Lomefloxacin hydrochloride . Linearity range (ng/mL) 1–1000 1–1000 1–1000 LOD (ng/mL) 0.293 0.278 0.268 LOQ (ng/mL) 0.971 0.926 0.894 Regression equation y* = bx** + a  Slope (b) 0.0192 0.0241 0.0035  Intercept (a) 0.1658 0.1713 0.0026 Coefficient of determination (r2) 0.9995 0.9995 0.9996 *Peak area ratio. **Concentration in ng/mL. Open in new tab Limits of detection and quantitation LOD and LOQ values were calculated for the proposed procedures based on the S/N ratio and the obtained results indicated the sensitivity of the proposed method as shown in Table I. Accuracy and precision (interday and intraday) The accuracy of the method was demonstrated as %R, and the precision of the method was demonstrated as %CV. Results are presented in Table II. Table II Intraday and interday accuracy and precision of cyclopentolate hydrochloride, phenylephrine hydrochloride and lomefloxacin hydrochloride determination by the proposed LC–MS/MS method Drug . Conc. (ng/mL) . Intraday . Interday . Found Conc.a ± SD Accuracy (%R) %CVb Found Conc.a ± SD Accuracy (%R) %CV Cyclopentolate hydrochloride 1 0.99 ± 0.016 98.51 1.616 0.99 ± 0.008 99.20 0.808 50 49.37 ± 0.325 98.73 0.658 50.58 ± 0.237 101.16 0.469 500 507.35 ± 6.903 101.47 1.361 494.30 ± 5.236 98.86 1.059 750 756.68 ± 12.230 100.89 1.616 761.55 ± 6.780 101.54 0.890 Phenylephrine hydrochloride 1 1.02 ± 0.019 101.67 1.863 0.99 ± 0.012 99.15 1.212 50 49.57 ± 0.508 99.14 1.025 49.12 ± 0.532 98.24 1.083 500 491.80 ± 4.475 98.36 0.910 503.60 ± 4.174 100.72 0.829 750 759.38 ± 9.322 101.25 1.228 758.18 ± 6.895 101.09 0.909 Lomefloxacin hydrochloride 1 0.99 ± 0.018 99.32 1.818 1.02 ± 0.015 101.65 1.471 50 49.49 ± 0.296 98.97 0.598 49.74 ± 0.422 99.48 0.848 500 490.65 ± 3.532 98.13 0.720 509.65 ± 3.150 101.93 0.618 750 757.20 ± 8.425 100.96 1.113 748.13 ± 9.865 99.75 1.319 Drug . Conc. (ng/mL) . Intraday . Interday . Found Conc.a ± SD Accuracy (%R) %CVb Found Conc.a ± SD Accuracy (%R) %CV Cyclopentolate hydrochloride 1 0.99 ± 0.016 98.51 1.616 0.99 ± 0.008 99.20 0.808 50 49.37 ± 0.325 98.73 0.658 50.58 ± 0.237 101.16 0.469 500 507.35 ± 6.903 101.47 1.361 494.30 ± 5.236 98.86 1.059 750 756.68 ± 12.230 100.89 1.616 761.55 ± 6.780 101.54 0.890 Phenylephrine hydrochloride 1 1.02 ± 0.019 101.67 1.863 0.99 ± 0.012 99.15 1.212 50 49.57 ± 0.508 99.14 1.025 49.12 ± 0.532 98.24 1.083 500 491.80 ± 4.475 98.36 0.910 503.60 ± 4.174 100.72 0.829 750 759.38 ± 9.322 101.25 1.228 758.18 ± 6.895 101.09 0.909 Lomefloxacin hydrochloride 1 0.99 ± 0.018 99.32 1.818 1.02 ± 0.015 101.65 1.471 50 49.49 ± 0.296 98.97 0.598 49.74 ± 0.422 99.48 0.848 500 490.65 ± 3.532 98.13 0.720 509.65 ± 3.150 101.93 0.618 750 757.20 ± 8.425 100.96 1.113 748.13 ± 9.865 99.75 1.319 aAverage of six determinations. Conc., concentration; SD, standard deviation. Open in new tab Table II Intraday and interday accuracy and precision of cyclopentolate hydrochloride, phenylephrine hydrochloride and lomefloxacin hydrochloride determination by the proposed LC–MS/MS method Drug . Conc. (ng/mL) . Intraday . Interday . Found Conc.a ± SD Accuracy (%R) %CVb Found Conc.a ± SD Accuracy (%R) %CV Cyclopentolate hydrochloride 1 0.99 ± 0.016 98.51 1.616 0.99 ± 0.008 99.20 0.808 50 49.37 ± 0.325 98.73 0.658 50.58 ± 0.237 101.16 0.469 500 507.35 ± 6.903 101.47 1.361 494.30 ± 5.236 98.86 1.059 750 756.68 ± 12.230 100.89 1.616 761.55 ± 6.780 101.54 0.890 Phenylephrine hydrochloride 1 1.02 ± 0.019 101.67 1.863 0.99 ± 0.012 99.15 1.212 50 49.57 ± 0.508 99.14 1.025 49.12 ± 0.532 98.24 1.083 500 491.80 ± 4.475 98.36 0.910 503.60 ± 4.174 100.72 0.829 750 759.38 ± 9.322 101.25 1.228 758.18 ± 6.895 101.09 0.909 Lomefloxacin hydrochloride 1 0.99 ± 0.018 99.32 1.818 1.02 ± 0.015 101.65 1.471 50 49.49 ± 0.296 98.97 0.598 49.74 ± 0.422 99.48 0.848 500 490.65 ± 3.532 98.13 0.720 509.65 ± 3.150 101.93 0.618 750 757.20 ± 8.425 100.96 1.113 748.13 ± 9.865 99.75 1.319 Drug . Conc. (ng/mL) . Intraday . Interday . Found Conc.a ± SD Accuracy (%R) %CVb Found Conc.a ± SD Accuracy (%R) %CV Cyclopentolate hydrochloride 1 0.99 ± 0.016 98.51 1.616 0.99 ± 0.008 99.20 0.808 50 49.37 ± 0.325 98.73 0.658 50.58 ± 0.237 101.16 0.469 500 507.35 ± 6.903 101.47 1.361 494.30 ± 5.236 98.86 1.059 750 756.68 ± 12.230 100.89 1.616 761.55 ± 6.780 101.54 0.890 Phenylephrine hydrochloride 1 1.02 ± 0.019 101.67 1.863 0.99 ± 0.012 99.15 1.212 50 49.57 ± 0.508 99.14 1.025 49.12 ± 0.532 98.24 1.083 500 491.80 ± 4.475 98.36 0.910 503.60 ± 4.174 100.72 0.829 750 759.38 ± 9.322 101.25 1.228 758.18 ± 6.895 101.09 0.909 Lomefloxacin hydrochloride 1 0.99 ± 0.018 99.32 1.818 1.02 ± 0.015 101.65 1.471 50 49.49 ± 0.296 98.97 0.598 49.74 ± 0.422 99.48 0.848 500 490.65 ± 3.532 98.13 0.720 509.65 ± 3.150 101.93 0.618 750 757.20 ± 8.425 100.96 1.113 748.13 ± 9.865 99.75 1.319 aAverage of six determinations. Conc., concentration; SD, standard deviation. Open in new tab Selectivity The selectivity of the method was checked by its ability to determine the analytes of interest in the matrix without any interference from other endogenous matrix components. It was confirmed by the absence of significant interference at the retention times for each drug or IS from six different batches of drug-free rabbit aqueous humor (blank aqueous humor) used for analysis. Representative chromatograms are shown in Figures 2–5. Extraction recovery and matrix effect The procedure proposed by Matuszewski et al. (19) was used for the determination of extraction recovery (%ER) and matrix effect (%ME) using three different sets of samples at three concentrations levels (LQC, MQC and HQC levels) for all analytes, whereas IS was at 1000 ng/mL. The ER was determined by calculating the ratios of the corresponding peak areas in the third set (C) to those in the second set (B) of samples, as follows: $$\%\boldsymbol{ER}=\frac{\boldsymbol{C}\left(\boldsymbol{mean}\kern0.17em \boldsymbol{peak}\kern0.17em \boldsymbol{area}\kern0.17em \boldsymbol{for}\kern0.17em \boldsymbol{standard}\kern0.17em \boldsymbol{spiked}\kern0.17em \boldsymbol{into}\kern0.17em \boldsymbol{aqueous}\kern0.17em \boldsymbol{humor}\kern0.17em \boldsymbol{before}\kern0.17em \boldsymbol{extraction}\right)}{\boldsymbol{B}\left(\boldsymbol{mean}\kern0.17em \boldsymbol{peak}\kern0.17em \boldsymbol{area}\kern0.17em \boldsymbol{for}\kern0.17em \boldsymbol{standard}\kern0.17em \boldsymbol{spiked}\kern0.17em \boldsymbol{into}\kern0.17em \boldsymbol{aqueous}\kern0.17em \boldsymbol{humor}\kern0.17em \boldsymbol{before}\kern0.17em \boldsymbol{extraction}\right)}\times 100 $$ Matrix effects were estimated by calculating the ratios of the corresponding peak areas in the second set (B) to those in the first set (A) of samples, as follows: $$\%\boldsymbol{ME}=\frac{\boldsymbol{B}\Big(\boldsymbol{mean}\kern0.17em \boldsymbol{peak}\kern0.17em \boldsymbol{area}\kern0.17em \boldsymbol{for}\kern0.17em \boldsymbol{standard}\kern0.17em \boldsymbol{spiked}\kern0.17em \boldsymbol{in}\boldsymbol{to}\kern0.17em \boldsymbol{aqueous}\kern0.17em \boldsymbol{humor}\;b\boldsymbol{efore}\kern0.17em \boldsymbol{extraction}\Big)}{\boldsymbol{A}\Big(\boldsymbol{mean}\kern0.17em \boldsymbol{peak}\kern0.17em \boldsymbol{area}\kern0.17em \boldsymbol{of}\kern0.17em \boldsymbol{neat}\kern0.17em \boldsymbol{standard}\kern0.17em \boldsymbol{in}\kern0.17em \boldsymbol{the}\kern0.17em \boldsymbol{mobile}\kern0.17em \boldsymbol{phase}\kern0.17em \boldsymbol{solution}\Big)}\times 100 $$ The extraction efficiency of the proposed method was assessed by the mean recovery for cited drugs in rabbit aqueous humor. Good recovery results were obtained and presented in Table III, indicating good extraction efficiency of the proposed method within the limits of variability and show that the matrix has no significant effect on ion suppression or enhancement or on extraction efficiency either of the analytes or the IS concerning the accuracy or the precision. As per the acceptance criteria, the recovery of the analytes do not need to be 100%, but the extent of recovery of an analyte should be consistent, precise and reproducible. Table III Extraction recovery and matrix effect on the determination of cyclopentolate hydrochloride, phenylephrine hydrochloride and lomefloxacin hydrochloride in aqueous humor by the proposed LC–MS/MS method Conc. level . Conc. (ng/mL) . Cyclopentolate hydrochloride . Phenylephrine hydrochloride . Lomefloxacin hydrochloride . Moxifloxacin hydrochloride (IS) (1000 ng/mL) . . . %ER . %ME . %ER . %ME . %ER . %ME . %ER . %ME . LQC 50 88.15 93.9 87.69 92.32 85.23 95.1 MQC 500 85.73 87.12 90.57 90.63 91.41 92.75 HQC 750 86.29 89.39 88.13 94.18 87.09 93.07 Meana ± SD 86.72 ± 1.267 90.14 ± 3.451 88.80 ± 1.551 92.38 ± 1.776 87.91 ± 3.171 93.64 ± 1.275 89.24 ± 2.671 92.26 ± 4.932 % Error 0.731 1.993 0.896 1.025 1.831 0.736 Conc. level . Conc. (ng/mL) . Cyclopentolate hydrochloride . Phenylephrine hydrochloride . Lomefloxacin hydrochloride . Moxifloxacin hydrochloride (IS) (1000 ng/mL) . . . %ER . %ME . %ER . %ME . %ER . %ME . %ER . %ME . LQC 50 88.15 93.9 87.69 92.32 85.23 95.1 MQC 500 85.73 87.12 90.57 90.63 91.41 92.75 HQC 750 86.29 89.39 88.13 94.18 87.09 93.07 Meana ± SD 86.72 ± 1.267 90.14 ± 3.451 88.80 ± 1.551 92.38 ± 1.776 87.91 ± 3.171 93.64 ± 1.275 89.24 ± 2.671 92.26 ± 4.932 % Error 0.731 1.993 0.896 1.025 1.831 0.736 aAverage of %ER and %ME at LQC, MQC and HQC each of six determinations. Open in new tab Table III Extraction recovery and matrix effect on the determination of cyclopentolate hydrochloride, phenylephrine hydrochloride and lomefloxacin hydrochloride in aqueous humor by the proposed LC–MS/MS method Conc. level . Conc. (ng/mL) . Cyclopentolate hydrochloride . Phenylephrine hydrochloride . Lomefloxacin hydrochloride . Moxifloxacin hydrochloride (IS) (1000 ng/mL) . . . %ER . %ME . %ER . %ME . %ER . %ME . %ER . %ME . LQC 50 88.15 93.9 87.69 92.32 85.23 95.1 MQC 500 85.73 87.12 90.57 90.63 91.41 92.75 HQC 750 86.29 89.39 88.13 94.18 87.09 93.07 Meana ± SD 86.72 ± 1.267 90.14 ± 3.451 88.80 ± 1.551 92.38 ± 1.776 87.91 ± 3.171 93.64 ± 1.275 89.24 ± 2.671 92.26 ± 4.932 % Error 0.731 1.993 0.896 1.025 1.831 0.736 Conc. level . Conc. (ng/mL) . Cyclopentolate hydrochloride . Phenylephrine hydrochloride . Lomefloxacin hydrochloride . Moxifloxacin hydrochloride (IS) (1000 ng/mL) . . . %ER . %ME . %ER . %ME . %ER . %ME . %ER . %ME . LQC 50 88.15 93.9 87.69 92.32 85.23 95.1 MQC 500 85.73 87.12 90.57 90.63 91.41 92.75 HQC 750 86.29 89.39 88.13 94.18 87.09 93.07 Meana ± SD 86.72 ± 1.267 90.14 ± 3.451 88.80 ± 1.551 92.38 ± 1.776 87.91 ± 3.171 93.64 ± 1.275 89.24 ± 2.671 92.26 ± 4.932 % Error 0.731 1.993 0.896 1.025 1.831 0.736 aAverage of %ER and %ME at LQC, MQC and HQC each of six determinations. Open in new tab Stability of analytes Short-term and long-term stability evaluations indicated that samples were stable >6 hours at room temperature and 35 days when stored in rabbit aqueous humor at −20°C. Three freeze and thaw cycles stability was also evaluated at LQC, MQC and HQC samples. As shown in Table IV, >85% of the analytes concentrations remained unchanged throughout all stability tests when compared with freshly prepared samples. Table IV Stability of cyclopentolate hydrochloride, phenylephrine hydrochloride and lomefloxacin hydrochloride determination in aqueous humor under different storage conditions by the proposed LC–MS/MS method Stability conditions . Conc. level . Conc.a (ng/mL) . %R ± SD . Cyclopentolate hydrochloride . Phenylephrine hydrochloride . Lomefloxacin hydrochloride . Short-term (6 hours at 20–25°C) LQC 50 88.43 ± 4.194 86.70 ± 5.323 92.28 ± 1.413 MQC 500 86.27 ± 3.453 90.56 ± 2.712 93.72 ± 1.356 HQC 750 90.15 ± 2.832 92.10 ± 6.128 91.37 ± 6.357 Long-term (35 days at −20°C) LQC 50 85.13 ± 5.727 85.67 ± 4.651 86.87 ± 2.685 MQC 500 85.69 ± 4.194 86.94 ± 3.870 90.41 ± 2.716 HQC 750 87.50 ± 1.216 89.20 ± 2.953 88.16 ± 3.236 Three freeze–thaw cycles LQC 50 86.24 ± 3.938 87.43 ± 1.057 90.98 ± 3.429 MQC 500 85.24 ± 6.786 89.71 ± 4.514 91.96 ± 1.357 HQC 750 87.62 ± 2.891 90.75 ± 1.442 89.10 ± 2.872 Stability conditions . Conc. level . Conc.a (ng/mL) . %R ± SD . Cyclopentolate hydrochloride . Phenylephrine hydrochloride . Lomefloxacin hydrochloride . Short-term (6 hours at 20–25°C) LQC 50 88.43 ± 4.194 86.70 ± 5.323 92.28 ± 1.413 MQC 500 86.27 ± 3.453 90.56 ± 2.712 93.72 ± 1.356 HQC 750 90.15 ± 2.832 92.10 ± 6.128 91.37 ± 6.357 Long-term (35 days at −20°C) LQC 50 85.13 ± 5.727 85.67 ± 4.651 86.87 ± 2.685 MQC 500 85.69 ± 4.194 86.94 ± 3.870 90.41 ± 2.716 HQC 750 87.50 ± 1.216 89.20 ± 2.953 88.16 ± 3.236 Three freeze–thaw cycles LQC 50 86.24 ± 3.938 87.43 ± 1.057 90.98 ± 3.429 MQC 500 85.24 ± 6.786 89.71 ± 4.514 91.96 ± 1.357 HQC 750 87.62 ± 2.891 90.75 ± 1.442 89.10 ± 2.872 aAverage of six determinations. Open in new tab Table IV Stability of cyclopentolate hydrochloride, phenylephrine hydrochloride and lomefloxacin hydrochloride determination in aqueous humor under different storage conditions by the proposed LC–MS/MS method Stability conditions . Conc. level . Conc.a (ng/mL) . %R ± SD . Cyclopentolate hydrochloride . Phenylephrine hydrochloride . Lomefloxacin hydrochloride . Short-term (6 hours at 20–25°C) LQC 50 88.43 ± 4.194 86.70 ± 5.323 92.28 ± 1.413 MQC 500 86.27 ± 3.453 90.56 ± 2.712 93.72 ± 1.356 HQC 750 90.15 ± 2.832 92.10 ± 6.128 91.37 ± 6.357 Long-term (35 days at −20°C) LQC 50 85.13 ± 5.727 85.67 ± 4.651 86.87 ± 2.685 MQC 500 85.69 ± 4.194 86.94 ± 3.870 90.41 ± 2.716 HQC 750 87.50 ± 1.216 89.20 ± 2.953 88.16 ± 3.236 Three freeze–thaw cycles LQC 50 86.24 ± 3.938 87.43 ± 1.057 90.98 ± 3.429 MQC 500 85.24 ± 6.786 89.71 ± 4.514 91.96 ± 1.357 HQC 750 87.62 ± 2.891 90.75 ± 1.442 89.10 ± 2.872 Stability conditions . Conc. level . Conc.a (ng/mL) . %R ± SD . Cyclopentolate hydrochloride . Phenylephrine hydrochloride . Lomefloxacin hydrochloride . Short-term (6 hours at 20–25°C) LQC 50 88.43 ± 4.194 86.70 ± 5.323 92.28 ± 1.413 MQC 500 86.27 ± 3.453 90.56 ± 2.712 93.72 ± 1.356 HQC 750 90.15 ± 2.832 92.10 ± 6.128 91.37 ± 6.357 Long-term (35 days at −20°C) LQC 50 85.13 ± 5.727 85.67 ± 4.651 86.87 ± 2.685 MQC 500 85.69 ± 4.194 86.94 ± 3.870 90.41 ± 2.716 HQC 750 87.50 ± 1.216 89.20 ± 2.953 88.16 ± 3.236 Three freeze–thaw cycles LQC 50 86.24 ± 3.938 87.43 ± 1.057 90.98 ± 3.429 MQC 500 85.24 ± 6.786 89.71 ± 4.514 91.96 ± 1.357 HQC 750 87.62 ± 2.891 90.75 ± 1.442 89.10 ± 2.872 aAverage of six determinations. Open in new tab Discussion In the present study, a sensitive, selective and fast analytical method for the simultaneous determination of cyclopentolate hydrochloride, phenylephrine hydrochloride and lomefloxacin hydrochloride using LC–MS/MS to achieve high sensitivity and selectivity for the detection and quantitation of cited drugs in rabbit aqueous humor with a fast and cheap extraction process, allowing the method to be of high use in evaluation of the cited drugs either alone or in combinations. For sample preparation, extraction methods including direct precipitation using acetonitrile and liquid–liquid extraction were studied. The use of liquid–liquid extraction has the advantage of effective deproteinization of the aqueous humor samples. The electrospray ionization interface parameters were optimized for all individual compounds in order to obtain the best instrumental conditions for the identification of target compounds. All the three compounds showed maximum sensitivity operating in the positive ionization mode. Conclusion A sensitive, selective, accurate and precise UPLC–MS/MS method was developed and validated for the simultaneous determination of drugs used as eye drops in cataract surgery, namely: cyclopentolate hydrochloride, phenylephrine hydrochloride and lomefloxacin hydrochloride in rabbit aqueous humor. The extraction procedure is simple with adequate recovery and the run time is short using isocratic mode of elution through UPLC system coupled with tandem mass spectrometric detection (LC–MS/MS). The proposed method was rapid enough to analyze large number of aqueous humor samples within a short period of time with high sensitivity and selectivity. The method is capable of estimation of the cited drugs accurately in rabbit aqueous humor either alone or in combinations. 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TI - Validated Liquid Chromatography–Tandem Mass Spectroscopic Method for Simultaneous Determination of Certain Drugs Used in Cataract Surgery in Rabbit Aqueous Humor
JF - Journal of Chromatographic Science
DO - 10.1093/chromsci/bmaa049
DA - 2020-09-29
UR - https://www.deepdyve.com/lp/oxford-university-press/validated-liquid-chromatography-tandem-mass-spectroscopic-method-for-t6ClxdhUbb
SP - 814
EP - 822
VL - 58
IS - 9
DP - DeepDyve
ER -