Determination of levocetirizine in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to a bioequivalence study.

We describe a liquid chromatography-tandem mass spectrometric method (LC-MS/MS) for levocetirizine quantification (I) in human plasma. Sample preparation was made using a fexofenadine (II) addition as internal standard (IS), liquid-liquid extraction using cold dichloromethane, and dissolving the final extract in acetonitrile. I and II (IS) were injected in a C18 column and the mobile phase composed of acetonitrile:water:formic acid (80.00:19.90:0.10, v/v/v) and monitored using positive electrospray source with tandem mass spectrometry analyses. The selected reaction monitoring (SRM) was set using precursor ion and product ion combinations of m/z 389>201 for I and m/z 502>467 for II. The limit of quantification and the dynamic range achieved were 0.5ng/mL and 0.5-500.0ng/mL. Validation results on linearity, specificity, accuracy, precision and stability, as well as its application to the analysis of plasma samples taken up to 48h after oral administration of 5mg of levocetirizine dichloridrate in healthy volunteers demonstrate its applicability to bioavailability studies.

Effect of Helicobacter pylori infection and acid blockade by lansoprazole on clarithromycin bioavailability.

The effect of proton pump inhibitors and Helicobacter pylori infection on the bioavailability of antibiotics is poorly understood. We determined the effects of 5-day oral administration of 60 mg lansoprazole on the bioavailability of clarithromycin in individuals with and without H. pylori infection. Thirteen H. pylori-infected and 10 non-infected healthy volunteers were enrolled in a study with an open-randomized two-period crossover design and a 21-day washout period between phases. Plasma concentrations of clarithromycin in subjects with and without lansoprazole pre-treatment were measured by liquid chromatography coupled to a tandem mass spectrometer. Clarithromycin Cmax and AUC0-10 h were significantly reduced after lansoprazole administration. In addition, lansoprazole treatment of the H. pylori-positive group resulted in a statistically significant greater reduction in Cmax (40 vs 15%) and AUC0-10 h (30 vs 10%) compared to lansoprazole-treated H. pylori-negative subjects. Thus, treatment with lansoprazole for 5 days reduced bioavailability of clarithromycin, irrespective of H. pylori status. This reduction, however, was even more pronounced in H. pylori-infected individuals.

Plasma hydroxy-metronidazole/metronidazole ratio in hepatitis C virus-induced liver disease.

It has been suggested that the measurement of metronidazole clearance is a sensitive method for evaluating liver function. The aim of this study was to evaluate the usefulness of plasma hydroxy-metronidazole/metronidazole ratios as indicators of dynamic liver function to detect changes resulting from the various forms of chronic hepatitis C virus (HCV) infection. A total of 139 individuals were studied: 14 healthy volunteers, 22 healthy, asymptomatic, consecutive anti-HCV-positive HCV-RNA negative subjects, 81 patients with chronic hepatitis C (49 with moderate/severe chronic hepatitis and 34 with mild hepatitis), and 20 patients with cirrhosis of the liver. HCV status was determined by the polymerase chain reaction. Plasma concentrations of metronidazole and its hydroxy-metabolite were measured by reverse-phase high-performance liquid chromatography with ultraviolet detection in a blood sample collected 10 min after the end of a metronidazole infusion. Anti-HCV-positive HCV-RNA-negative individuals demonstrated a significantly reduced capacity to metabolize intravenously infused metronidazole compared to healthy individuals (0.0478 +/- 0.0044 vs 0.0742 +/- 0.0232). Liver cirrhosis patients also had a reduced plasma hydroxy-metronidazole/metronidazole ratio when compared to the other groups of anti-HCV-positive individuals (0.0300 +/- 0.0032 vs 0.0438 +/- 0.0027 (moderate/severe chronic hepatitis) vs 0.0455 +/- 0.0026 (mild chronic hepatitis) and vs 0.0478 +/- 0.0044 (anti-HCV-positive, HCV-RNA-negative individuals)). These results suggest an impairment of the metronidazole metabolizing system induced by HCV infection that lasts after viral clearance. In those patients with chronic hepatitis C, this impairment is paralleled by progression of the disease to liver cirrhosis.

Amoxicillin and ampicillin are not transferred to gastric juice irrespective of Helicobacter pylori status or acid blockade by omeprazole.

BACKGROUND: The effects of proton pump inhibitors and Helicobacter pylori infection on the distribution of drugs used for the eradication of the bacteria are poorly understood. AIM: The aim of this study was to investigate the effects of a 7-day administration of 20 mg of omeprazole on the pharmacokinetics of amoxicillin and ampicillin in the plasma, saliva and gastric juice of individuals with and without H. pylori infection. METHODS: Fifty-four healthy volunteers without endoscopic lesions were enrolled. Twenty-six volunteers were included in the amoxicillin study and 28 individuals in the ampicillin study. Each study had an open randomized two-period crossover design and a 21-day washout period between phases. Plasma, saliva and gastric juice concentrations of amoxicillin and ampicillin in subjects with and without omeprazole pre-treatment were measured by reversed-phase HPLC using UV detection. RESULTS: Neither pre-treatment with omeprazole nor H. pylori infection interfered with the plasma bioavailability of amoxicillin or ampicillin, as assessed by the AUC0-2 h. Neither ampicillin nor amoxicillin were detected in saliva or gastric juice in any study phase. CONCLUSION: Short-term treatment with omeprazole does not interfere with the pharmacokinetics of amoxicillin or ampicillin. Our results also exclude the presence of a transfer mechanism for amoxicillin or ampicillin from the plasma to the gastric lumen.

Determination of amoxycillin in human plasma by direct injection and coupled-column high-performance liquid chromatography.

This work reports the use of multidimensional HPLC by coupling a restricted access medium (RAM) bovine serum albumin (BSA) octadecyl column (100 x 4.6 mm I.D., 10 microm particle size and 120 A pore size) to an octadecyl Hypersil column (150 x 4.6 mm I.D., 5 microm particle size and 120 A pore size) to the analysis of amoxycillin in human plasma by direct injection. Ion pairing was necessary to extract amoxycillin with good recovery from the plasma proteins. To prepare the spiked samples, aliquots (60 microl) of the appropriated standard solutions were added to each culture tube containing an 180 microl of plasma and a solution of 0.30 mM tetrabuthylammonium phosphate (60 microl). They were vortexed for 15 s and then 290 microl were transferred to autosampler vials. Aliquots (250 microl) of the spiked plasma samples were injected to a column-switching HPLC system. An analysis time of 25 min with no time spent on sample preparation was achieved. The developed method showed good selectivity, sensitivity, accuracy and precision for direct analysis of this polar low wavelength ultraviolet absorption antibiotic using only 180 microl of human plasma. The validated method proved to be reliable and sensitive for the determination of amoxycillin in plasma samples of five healthy volunteers to whom test and reference formulations were administered as an oral dose (500 mg).

An enantioselective assay for (+/-)-modafinil.

Enantiomers of modafinil, (diphenylmethyl)sulphinyl-2-acetamide, were separated by an amylose tris[(S)-1-phenylethylcarbamate] chiral stationary phase, using acetonitrile: water (25:75 v/v) as mobile phase, with excellent selectivity (alpha=1.4) and resolution factors (R(s)=1.5). The assay involved a solid-phase extraction of the enantiomers of modafinil from plasma using a C(18) Cartridge. A good linear relationship was obtained in the concentration of 0.15-3 microg ml(-1) for each enantiomer. The method developed is sufficiently accurate and precise to be used for clinical samples and has a good selectivity with the two main circulating metabolites: the (diphenylmethyl)sulphinyl-2-acetic acid and (diphenylmethyl)sulphonyl-2-acetamide. The use of a polysaccharide-based column on multimodal elution was explored in developing the method.

Development and validation of a selective and robust LC-MS/MS method for quantifying amlodipine in human plasma.

A liquid chromatographic-tandem mass spectrometric method (LC-MS/MS) for quantifying amlodipine in human plasma was developed and validated. Sample preparation was based on liquid-liquid extraction using NaOH and a mixture of ethyl acetate/hexane (80/20; v/v). Chromatography was performed on a C-18 analytical column and the retention times were 1.9 and 3.0 min for amlodipine and nimodipine (internal standard), respectively. The ionization was optimized using ESI(+) and enhanced selectivity was achieved using tandem mass spectrometric analysis via two MRM functions, 409 --> 238 and 418 --> 343 for amlodipine and nimodipine. The calibration curve ranged from 0.2 to 20.0 ng/mL. The inter-day precision and accuracy and the relative standard deviation (RSD) were <15%. The analyte was shown to be stable over the time-scale of the whole procedure. The robustness of the method was demonstrated by the good reproducibility of the results obtained during the analysis of clinical samples.

Effect of acid secretion blockade by omeprazole on the relative bioavailability of orally administered furazolidone in healthy volunteers.

AIMS: The administration of omeprazole may interfere with the absorption of orally administered drugs by reducing gastric pH and hence tablet dissolution. The aim of this study was to investigate the effects of a 5 day administration of omeprazole on the pharmacokinetics of furazolidone. METHODS: Eighteen healthy (nine male and nine female) volunteers were selected. The study had an open randomized two-period crossover design with a 21 day washout period between the phases. Serum concentrations of furazolidone were measured by reversed-phase h.p.l.c. with ultraviolet detection. RESULTS: Administration of omeprazole caused a significant reduction of Cmax [0.34 microg x ml(-1) (range 0.25-0.43) vs 0.24 microg x ml(-1) (range 0.15-0.34)] with no significant delay in absorption tmax [2.5 h (range 1.85-3.0) vs 2.4 h (range 2.06-2.71)]. CONCLUSIONS: Furazolidone was rapidly absorbed after oral administration. Short-term treatment with omeprazole did alter the relative bioavailability of this drug, probably through an effect on absorption kinetics or first-pass metabolism.

Transfer of clarithromycin to gastric juice is enhanced by omeprazole in Helicobacter pylori-infected individuals.

BACKGROUND: The effects of proton-pump inhibitors and Helicobacter pylori infection on the distribution of drugs employed for the eradication of H. pylori are poorly understood. The aim of this study was to investigate the effects of a 7-day oral administration of 20 mg omeprazole on the distribution of clarithromycin in the gastric juice of individuals with H. pylori infection. METHODS: Eighteen H. pylori-infected dyspeptic male volunteers without endoscopic lesions were enrolled in a study with an open, randomized, two-period crossover design and a 21-day washout period between phases. Plasma and gastric juice concentrations of clarithromycin in subjects with and without omeprazole pretreatment were measured by means of liquid chromatography coupled to tandem mass spectrometry. RESULTS: The maximum concentration of clarithromycin (Cmax) and the area under the time-concentration curve from 0 to 2 h (AUC0-2h) were significantly higher in gastric juice than in plasma. Omeprazole treatment further augmented clarithromycin Cmax and AUC0-2h in gastric juice approximately 2-fold (P < 0.05). CONCLUSIONS: Short-term treatment with omeprazole in H. pylori-positive volunteers increases the amount of clarithromycin transferred to the gastric juice, confirming a synergism between these drugs. Our results suggest the presence of an active transport mechanism for clarithromycin from plasma to the gastric lumen, which is influenced by omeprazole.

Transfer of metronidazole to gastric juice: impact of Helicobacter pylori infection and omeprazole.

BACKGROUND: The effects of Helicobacter pylori infection associated with inhibition of gastric acid secretion on the distribution of medications used for H. pylori eradication are poorly understood. The aim of this study was to investigate the effects of a 7-day administration of 20 mg omeprazole on the transfer of metronidazole from plasma to the gastric juice of individuals with and without H. pylori infection. METHODS: Fourteen H. pylori-positive and 14 H. pylori-negative male volunteers were enrolled in a study with an open, randomized, two-period crossover design with a 21-day washout period between phases. Plasma, salivary, and gastric juice concentrations of metronidazole in subjects with and without omeprazole treatment were measured with reversed-phase high-performance liquid chromatography/liquid chromatography. RESULTS: Metronidazole peak concentration (Cmax) was similar in plasma and saliva and was approximately threefold higher in gastric juice in all groups. Omeprazole treatment increased gastric pH and did not affect metronidazole Cmax or the time required for this to be reached (tmax) in plasma, saliva, or gastric juice. However, omeprazole significantly reduced metronidazole transfer from plasma to gastric juice in H. pylori-positive but not H. pylori-negative subjects, as shown by statistical analysis of AUC(0-2 h). CONCLUSION: Short-term treatment with omeprazole in H. pylori- positive volunteers reduces the amount of metronidazole transferred from plasma to gastric juice. This seems to occur in a pH-independent form.

Effect of Helicobacter pylori infection and acid blockade by lansoprazole on clarithromycin bioavailability

The effect of proton pump inhibitors and Helicobacter pylori infection on the bioavailability of antibiotics is poorly understood. We determined the effects of 5-day oral administration of 60 mg lansoprazole on the bioavailability of clarithromycin in individuals with and without H. pylori infection. Thirteen H. pylori-infected and 10 non-infected healthy volunteers were enrolled in a study with an open-randomized two-period crossover design and a 21-day washout period between phases. Plasma concentrations of clarithromycin in subjects with and without lansoprazole pre-treatment were measured by liquid chromatography coupled to a tandem mass spectrometer. Clarithromycin Cmax and AUC0-10 h were significantly reduced after lansoprazole administration. In addition, lansoprazole treatment of the H. pylori-positive group resulted in a statistically significant greater reduction in Cmax (40 vs 15 percent) and AUC0-10 h (30 vs 10 percent) compared to lansoprazole-treated H. pylori-negative subjects. Thus, treatment with lansoprazole for 5 days reduced bioavailability of clarithromycin, irrespective of H. pylori status. This reduction, however, was even more pronounced in H. pylori-infected individuals.

Plasma hydroxy-metronidazole/ metronidazole ratio in hepatitis C virus-induced liver disease

It has been suggested that the measurement of metronidazole clearance is a sensitive method for evaluating liver function. The aim of this study was to evaluate the usefulness of plasma hydroxy-metronidazole/metronidazole ratios as indicators of dynamic liver function to detect changes resulting from the various forms of chronic hepatitis C virus (HCV) infection. A total of 139 individuals were studied: 14 healthy volunteers, 22 healthy, asymptomatic, consecutive anti-HCV-positive HCV-RNA negative subjects, 81 patients with chronic hepatitis C (49 with moderate/severe chronic hepatitis and 34 with mild hepatitis), and 20 patients with cirrhosis of the liver. HCV status was determined by the polymerase chain reaction. Plasma concentrations of metronidazole and its hydroxy-metabolite were measured by reverse-phase high-performance liquid chromatography with ultraviolet detection in a blood sample collected 10 min after the end of a metronidazole infusion. Anti-HCV-positive HCV-RNA-negative individuals demonstrated a significantly reduced capacity to metabolize intravenously infused metronidazole compared to healthy individuals (0.0478 ± 0.0044 vs 0.0742 ± 0.0232). Liver cirrhosis patients also had a reduced plasma hydroxy-metronidazole/metronidazole ratio when compared to the other groups of anti-HCV-positive individuals (0.0300 ± 0.0032 vs 0.0438 ± 0.0027 (moderate/severe chronic hepatitis) vs 0.0455 ± 0.0026 (mild chronic hepatitis) and vs 0.0478 ± 0.0044 (anti-HCV-positive, HCV-RNA-negative individuals)). These results suggest an impairment of the metronidazole metabolizing system induced by HCV infection that lasts after viral clearance. In those patients with chronic hepatitis C, this impairment is paralleled by progression of the disease to liver cirrhosis.