Mohammed E. Abdel-Hamid

Determination of diclofenac sodium, flufenamic acid, indomethacin and ketoprofen by LC-APCI-MS.

A sensitive, selective and accurate high-performance liquid chromatography-mass spectrometry (LC-MS) assay for the determination of selected non-steroidal anti-inflammatory drugs (NSAIDs), namely diclofenac sodium (DIC), flufenamic acid (FLU), indomethacin (IND) and ketoprofen (KET), either individually or in mixtures, was developed. The examined drugs were injected onto Shim-pack GLC-CN column and were eluted with a mobile phase consisting of acetonitrile and 20 mM ammonium acetate solution (5:1 v/v)/pH 7.4 at a flow rate l ml min(-1). The mass spectrometer, operated in the single ion monitoring mode, was programmed to admit the negative ions [M-H] at m/z 295.9 (DIC), 280.1 (FLU), 355.8 (IND) and 252.9 (KET), respectively. The calibration curves were linear (r > or = 0.9993) over the concentration range 50-300 ng ml(-1) (FLU, DIC) and 100-500 ng ml(-1) (KET, IND) with detection limits of 0.5-4.0 ng. The mean predicted concentrations for the analytes were in the range -5.9 and 5.2% of the nominal concentrations. Within-day and between-day precision were in the range of 0.8-9.1% of the R.S.D. Mean recovery percentages of the individual compounds from laboratory-made mixtures and pharmaceutical formulations were (99.5-101.5%) and (100.6-102.2%), respectively.

Synthesis and antibacterial activity of novel 5-(4-methyl-1H-1,2,3-triazole) methyl oxazolidinones.

A series of 5-(4-methyl-1,2,3-triazole)methyl oxazolidinones were synthesized and tested for their antibacterial activity against a panel of Gram-positive and Gram-negative clinical isolates in comparison with linezolid and vancomycin. Most of the compounds demonstrated strong to moderate in vitro antibacterial activity against susceptible and resistant Gram-positive pathogenic bacteria. Antibacterial activity varied with substitutions at the phenyl C4 position with bulky alkylcarbonyl and alkoxycarbonyl substitutions on the piperazine N4 being detrimental to antibacterial activity. Whereas the presence of the 4-methyl-1,2,3-triazole moiety in the acyl-piperazine containing analogs resulted in increased protein binding, and decreased antibacterial activity particularly against Streptococcus pneumoniae strains.

Comparative LC–MS and HPLC analyses of selected antiepileptics and beta-blocking drugs

A highly sensitive and specific assay procedure based on the combination of liquid chromatography and mass spectrometry (LC-MS) has been developed for the quantitative analysis of selected antiepileptics (carbamazepine and phenytoin) and beta-blocking drugs (acebutolol, atenolol, pindolol and propranolol) using APCI as an ionization process. The measured concentration range was 100-300 ng ml-1 for all drugs except phenytoin (0.5-1.5 micrograms ml-1). Analysis was based on direct injection of methanolic solutions of drugs into the mass spectrometer with the subsequent elution with a mobile phase consisting of methanol and 1% acetic acid solution (4:1) at a flow rate 1 ml min-1. The mass spectrometer was programmed to permit detection and determination of either fragment or molecular ions of carbamazepine, phenytoin, acebutolol, atenolol, pindolol and propranolol at m/e 194.3, 252.9, 337.2, 267.1, 249.1 and 260.1, respectively. The recorded chromatograms exhibited well-resolved peaks at retention times < 1 min. The peak area was correlated linearly to the drug concentration. Intraday precision gave relative standard deviations in the range 1.75-4.02%. Compared to HPLC, the described LC-MS was faster, more sensitive and specific. Unlike HPLC, LC-MS could be applied to analyze incompletely resolved mixtures. The absolute detection limits for LC-MS and HPLC were 0.2-0.5 and 10-25 ng, respectively. Recovery studies of the investigated compounds in pharmaceutical products using LC-MS and HPLC gave mean percentages of 97.5-102.0 and 98.4-103.3, respectively. Statistical analysis of the data using t- and F-tests showed insignificant differences between both methods for the analysis of carbamazepine, phenytoin, acebutolol and atenolol in pharmaceutical formulations. However, LC-MS gave more accurate results than HPLC for determination of pindolol in tablets. Propranolol could only be determined in tablets using LC-MS.

Liquid chromatographic-mass spectrometric determination of celecoxib in plasma using single-ion monitoring and its use in clinical pharmacokinetics.

Celecoxib is a cyclooxygenase-2 specific inhibitor, that has been recently and intensively prescribed as an anti-inflammatory drug in rheumatic osteoarthritis. A robust, highly reliable and reproducible liquid chromatographic-mass spectrometric assay is developed for the determination of celecoxib in human plasma using sulindac as an internal standard. The run cycle-time is <4 min. The assay method involved extraction of the analytes from plasma samples at pH 5 with ethyl acetate and evaporation of the organic layer. The reconstituted solution of the residue was injected onto a Shim Pack GLC-CN, C18 column and chromatographed with a mobile phase comprised of acetonitrile-1% acetic acid solution (4:1) at a flow-rate of 1 ml/min. The mass spectrometer (LCQ Finnigan Mat) was programmed in the positive single-ion monitoring mode to permit the detection and quantitation of the molecular ions of celecoxib and sulindac at m/z 382 and 357, respectively. The peak area ratio of celecoxib/sulindac and concentration are linear (r2>0.994) over the concentration range 50-1000 ng/ml with a lowest detection limit of 20 ng/ml of celecoxib. Within- and between-day precision are within 1.58-4.0% relative standard deviation and the accuracy is 99.4-107.3% deviation of the nominal concentrations. The relative recoveries of celecoxib from human plasma ranged from 102.4 to 103.3% indicating the suitability of the method for the extraction of celecoxib and I.S. from plasma samples. The validated LC-MS method has been utilized to establish various pharmacokinetic parameters of celecoxib following a single oral dose administration of celecoxib capsules in two selected volunteers.

Determination of linezolid in human plasma by LC-MS-MS

A rapid, sensitive and selective LC-atmospheric pressure-chemical ionization-MS-MS method for the determination of the new antimicrobial agent, linezolid, in human plasma using selected reaction monitoring (SRM) was developed. Linezolid and the internal standard were extracted from the biological samples by solid phase extraction (SPE) and analyzed on a reversed-phase Shim Pack CLC-CN, C18 column with the mobile phase of acetonitrile and 20 mM ammonium acetate solution (4 + 1 v/v). Detection was accomplished using an LCQ mass spectrometer (Finnigan), which was programmed in positive MS-MS mode to permit measurement of the fragment ions of linezolid and internal standard at m/z 296.2 and 223.2, respectively. The assay run-time was less than 3.5 min. Quantitative analysis was based on peak area ratio of linezolid to the internal standard. Calibration plots were established over the concentration range of 0.1-20 micrograms ml-1 of linezolid with the lowest detection limit of 0.05 microgram ml-1 using 10 microliters sample volume. The SPE technique quantitatively recovered linezolid and the internal standard from the plasma samples at a percentage range of 89.1-93.7%. Determination of control samples of linezolid in plasma validated the LC-MS-MS-SRM method. Intra-assay and inter-assay precision were in the range of 5.1-11.4% relative standard deviation, whereas the intra- and inter-accuracy were in the range of 97.5-114.0% of the nominal concentrations of linezolid added. The data confirmed that the plasma samples of linezolid were stable at room temperature and when stored at -20 degrees C for at least 10 d. The developed LC-MS-MS-SRM method is recommended for the determination of linezolid in human plasma.

High-performance liquid chromatography-mass spectrometric analysis of furosemide in plasma and its use in pharmacokinetic studies.

This study presents a rapid, specific and sensitive high-performance liquid chromatography-mass spectrometric (LC-MS) assay for the determination of furosemide in human plasma using diclofenac as an internal standard (IS). Both compounds were extracted from human plasma with ethyl acetate at pH 1 and were chromatographed using Shim-Pack GLC-CN column and a mobile phase consisting of acetonitrile and 20 mM ammonium acetate buffer solution pH 7, 4:1 (v/v) at a flow rate 1 ml min(-1). Furosemide and IS were detected by mass spectrometer operated in the negative single ion monitoring mode using APCI as an ionization process at m/z 329.2 and 294.1, respectively. The assay linearity of furosemide was confirmed over the range 50-2,000 ng ml(-1). Detection limit for furosemide in plasma was 10 ng ml(-1). The selected concentration range corresponds well with the plasma concentrations of furosemide for pharmacokinetic study. Intraday and interday relative standard deviations were 1.3-4.7 and 2.7-11.5%, respectively. The extraction recovery percentages of furosemide and IS from plasma were in the range 89.3-97.1%. The developed LC-MS procedure was applied for the determination of the pharmacokinetic parameters of furosemide after an oral administration of tablet formulation (40 mg) to two healthy male volunteers. The calculated parameters were in good agreement with the reported values.

Development of LC–MS method for determination of ursolic acid: application to the analysis of ursolic acid in Staphylea holocarpa Hemsl.

Ursolic acid is a hydroxy pentacyclic triterpene, which has a chemoprotective activity in human. A reliable and reproducible liquid chromatography-mass spectrometric assay (LC-MS) was developed for the determination of ursolic acid in laboratory-made mixtures and in leaves and twigs extracts of Staphylea holocarpa Hemsl. The methanolic solution of the extracted ursolic acid was chromatographically analyzed using Shim Pack CLC-CN, C18 (150 x 6 mm, 5 mu) column and a mobile consisting of methanol-1% acetic acid solution (4:1) at a flow rate of 1.0 ml min(-1). The mass spectrometer (LCQ-Finnigan) was programmed in the positive single ion monitoring (SIM) to permit detection and quantitation of ursolic acid in MS-SIM mode at m/z 439.2, 411.2 and 390.9. Linear correlation (r > 0.99) of the peak area and the concentration of ursolic acid over the concentration range 0.25-10 microg ml(-1) was obtained. The relative standard deviation (%R.S.D.) and percentage deviation from the nominal concentrations (%DEV) were found to be 3.03-3.59% and -4.5 to +6.2%, respectively. Analysis of laboratory-made mixtures containing known concentrations of ursolic acid, as quality control samples, gave a mean recovery percentage of 97.8%. Application of the proposed method for the analysis of leaves and twigs extracts of S. holocarpa Hemsl. gave mean percentage contents of ursolic acid of 0.95 and 0.25%, respectively.

LC-MS/MS determination of Synercid® injections

Synercid is a combination of two semisynthetic pristinamycin derivatives, quinupristin and dalfopristin in 30:70 (w/w) ratio. A rapid and specific high-performance liquid chromatography-mass spectrometry was developed for the determination of quinupristin and dalfopristin using positive electrospray tandem mass spectrometry (+ESI-MS/MS). Multiple reaction monitoring transitions at 1023.05>134.34 and 691.87>166.26 were selected for the quantitation of quinupristin and dalfopristin, respectively. The assay run cycle-time was approximately 2.0 min injection-to-injection. The assay was linear up to concentration of 4000 ng x ml(-1) quinupristin and 1920 ng x ml(-1) dalfopristin. The lowest limits of quantitation of quinupristin and dalfopristin were found to be 1000 and 480 ng x ml(-1), respectively. Quantitation was based on peak area measurement of quinupristin and dalfopristin using weighed linear regression. Linear relationships with correlation coefficients (r>0.99) were automatically computed for both constituents by MASSLYNX quantify program. The ratio of the slopes of the calibration curves of quinupristin and dalfopristin was found to be 0.425, which matches the nominal ratio composition of the antimicrobial compounds in Synercid. The %RSD ranges were 2.3-4.0% for dalfopristin and 1.3-4.2% for quinupristin, whereas the %DEV ranges were (-7.5+3.7) and (-1.2+9.1%), respectively, indicating appropriate precision and accuracy. Recoveries of 99.5-103.8% and 97.8-99.0% of quinupristin and dalfopristin, respectively, were computed from Synercid injection. The described method is recommended for rapid determination of the contents and for tracking the stability and compatibility of quinupristin and dalfopristin in Synercid injection.

LC-MS ANALYSIS OF SELECTED SULFUR-CONTAINING NON-STEROID ANTI-INFLAMMATORY AGENTS: APPLICATIONS TO PHARMACEUTICAL PRODUCTS

A rapid, specific, sensitive, and reproducible high performance liquid chromatography-mass spectrometric assay has been developed for the quantitation of five sulfur-containing non-steroid anti-inflammatory drugs (NSAIDs), namely, celecoxib (Cel), piroxicam (Per), rofecoxib (Rof), sulindac (Sul), and tenoxicam (Ten), in available tablets and capsules. The examined compounds were extracted from the dosage forms with methanol and chromatographed on a Shim-Pack column using a mobile phase of acetonitrile and 1% acetic acid solution in a ratio 4:1 (Cel, Per, Sul, Ten) or acetonitrile and 20 mM ammonium acetate buffer solution (4:1) (Rof). The analytes were determined by an ion-trap mass spectrometer (Finnigan Mat) using APCI as an ionization process. The eluted compounds were detected either in the positive single ion monitoring mode at m/z 382.0, 332.0, 357.0, 338.4 (Cel, Per, Sul, Ten), or in the negative single ion monitoring mode at m/z 313.2 (Rof). Linear correlations of the peak area and concentration were confirmed for all compounds over the concentration ranges 0.25–1.0 μg /mL (Cel), 0.5–1.5 μg/mL (Per), 0.25–1.0 μg/mL (Sul), 1.0–2.0 μg/mL (Ten), and 0.1–1.0 μg/mL (Rof). The high specificity of the method was elucidated by analyzing mixtures of piroxicam/tenoxicam and celecoxib/sulindac. The developed method was successfully applied to determine the examined drugs in marketed pharmaceutical products.

Simultaneous Quantification of Doxorubicin, Lorazepam, Metoclopramide, Ondansetron, and Ranitidine in Mixtures by Liquid Chromatography‐Tandem Mass Spectrometry

Abstract A simple and rapid LC‐MS/MS method for simultaneous quantification of a mixture of the antineoplastic agent doxorubicin (Dox) and selected emesis suppressants, namely, lorazepam (Lor), metoclopramide (Met), ondansetron (Ond), and ranitidine (Ran), was developed. 4‐Phenyl‐4‐aminobutanoic acid was used as the internal standard (I.S.). The analytes were analyzed on an Xterra™ MS, C18, 2.5 µm (2.1 mm × 30 mm) column using 70% aqueous methanol containing 0.1% formic acid. The investigated compounds were detected by tandem mass spectrometry (MS/MS) using a positive electrospray ionization mode. Multiple reaction monitoring transitions at m/z 544 > 361, 323 > 277, 301 > 228, 294 > 169, 315 > 176 and 180 > 163 (I.S.) were selected for simultaneous quantitation of Dox, Lor, Met, Ond, and Ran by an I.S. method. Calibration curves were constructed over the concentration ranges 10–200 ng mL−1 (Dox, Lor), 8–80 ng mL−1 (Ond), Met and Ran (1–10 ng mL−1). The coefficient of determination ranged from 0.9953 to 0.9998 with a limit of detection of ∼1 ng mL−1 for all analytes. The overall percentage standard deviation and percentage deviation were <10% indicating good precision and accuracy. The developed LC‐MS/MS method quantitatively recovered doxorubicin and the co‐admixed compounds in the range 98.1%–103.5%. The data suggest the utility of the developed LC‐MS/MS procedure for simultaneous routine analysis of mixtures of Dox and the emesis suppressants, Lor, Met, Ond, and Ran in I.V. infusions in clinics.