Simultaneous determination of seven penicillins in muscle, liver and kidney tissues from cattle and pigs by a multiresidue high-performance liquid chromatographic method

A high-performance liquid chromatographic (HPLC) method based on solid-phase extraction (SPE) was developed for determination of amoxicillin, penicillin G (benzylpenicillin), ampicillin, oxacillin, cloxacillin, nafcillin and dicloxacillin in muscle, liver and kidney tissues of pigs and cattle. The compounds were extracted in aqueous solution by precipitation of organic materials with a mixture of sulphuric acid and sodium tungstate. The extract was cleaned up by SPE on a divinylbenzene-co-N-vinylpyrrolidone polymeric sorbent. Further clean-up was performed by liquid–liquid partition with diethyl ether. The extract was derivatised with benzoic anhydride and 1,2,4-triazole mercury (II) reagent. Chromatography was performed by reversed-phase gradient HPLC on a C₁₈ column with ultraviolet detection at 323 nm. The limits of detection estimated by a conservative model were in the range 8.9–11.1 μg/kg for amoxicillin, penicillin G, ampicillin, oxacillin, cloxacillin and nafcillin and 18.3–20.9 μg/kg for dicloxacillin. The mean recovery range was 66–77% for amoxicillin, 73–75% for penicillin G, 81–82% for ampicillin, 73–76% for oxacillin, 74–75% for cloxacillin, 66–72% for nafcillin and 58–65% for dicloxacillin.     

Comparison of amoxicillin and ampicillin in single-dose oral treatment of males with gonococcal urethritis

Amoxicillin in single oral doses of 2.0 g, 2.0 g plus 1.0 g probenecid, or 3.0 g was compared with ampicillin 3.5 g plus 1.0 g probenecid in the treatment of 203 males with uncomplicated acute gonococcal urethritis. Cure rates above 95% were produced by all treatments except the 2.0-g amoxicillin dose, which cured 89% of patients. Of 198 pretreatment gonococcus isolates tested by an agar dilution technique for susceptibility to penicillin G, ampicillin and amoxicillin, over 50% showed relative resistance (MIC > 0.06 μg/ml) to the antibiotics. However, amoxicillin was somewhat more active against isolates showing considerable resistance (MIC ≥ 1.0 μg/ml) to penicillin G or ampicillin. Adverse effects of amoxicillin were few: two patients reported transient nausea and six noted short-lived diarrhea. No hypersensitivity reactions were observed.     

Flucloxacillin,a New Isoxazolyl Penicillin,Compared with Oxacillin,Cloxacillin, and Dicloxacillin

Flucloxacillin, a new isoxazole penicillin, is active against penicillinase-producing strains of Staphylococcus aureus and is well absorbed in man after oral and intramuscular administration. Compared with isoxazole penicillins in current clinical use—namely, oxacillin, cloxacillin, and dicloxacillin—flucloxacillin has proved as active against Gram-positive cocci, including penicillin-resistant staphylococci. The extent of binding of flucloxacillin to the protein of human serum was similar to that of oxacillin and cloxacillin and less than that of dicloxacillin. In man flucloxacillin given orally produced total and free serum levels higher than those obtained with oxacillin and cloxacillin; total serum levels similar to those of dicloxacillin, and free levels greater than those of dicloxacillin. Similarly, after intramuscular injection the free serum levels of flucloxacillin were higher than those of oxacillin, cloxacillin, and dicloxacillin.     

Determination of penicillin G in bovine plasma by high-performance liquid chromatography after pre-column derivatization

A simple, selective, and sensitive liquid chromatographic method with ultraviolet detection was developed for the analysis of penicillin G in bovine plasma. The assay utilizes a simple extraction of penicillin G from plasma (with a known amount of penicillin V added as internal standard) with water, dilute sulphuric acid and sodium tungstate solutions, followed by concentration on a conditioned C₁₈ solid-phase extraction column. After elution with 500 μl of elution solution, the penicillins are derivatized with 500 μl of 1,2,4-triazole—mercuric chloride solution at 65°C for 30 min. The penicillin—mercury mercaptide complexes are separated by reversed-phase liquid chromatography on a C₁₈ column. The method, which has a detection limit of 5 ng/ml (ppb) in bovine plasma, was used to quantitatively measure the concentrations of penicillin G in plasma of steers at a series of intervals after the intramuscular administration of a commercial formulation of procaine penicillin G.     

High-performance liquid chromatographic analysis of amoxicillin in human and chinchilla plasma, middle ear fluid and whole blood

We extended the application of a sensitive high-performance liquid chromatography assay of amoxicillin developed in this laboratory for human plasma and middle ear fluid (MEF) to other sample matrices including chinchilla plasma or MEF and human and chinchilla whole blood with minor modification and validated the limit of quantitation at 0.25 μg/ml with a 50-μl sample size for human and chinchilla plasmas or MEFs. Amoxicillin and cefadroxil, the internal standard, were extracted from 50 μl of the samples with Bond Elut C₁₈ cartridges. The extract was analyzed on a Keystone MOS Hypersil-1 (C₈) column with UV detection at 210 nm. The mobile phase was 6% acetonitrile in 5 mM phosphate buffer, pH 6.5 and 5 mM tetrabutylammonium. The within-day coefficients of variation were 2.7–9.9 (n=4) and 1.7–7.2% (n=3) for chinchilla plasma and MEF samples, respectively; 2.8–8.1% (n=3) and 2.9–4.7% (n=3) for human and chinchilla whole blood, respectively. An alternative mobile phase composition for chinchilla plasma and MEF samples reduced the analysis time significantly.     

Sensitive and convenient high-performance liquid chromatographic method for the determination of mitomycin C in human plasma

An improved high-performance liquid chromatographic assay for the cytostatic drug mitomycin C in plasma is presented. The principal steps are precipitation of plasma proteins with acetonitrile, lyophilization of the supernatant and reversed-phase chromatography on a Hypersil ODS 5 μm column with 0.01 M NaH₂PO₄ buffer (pH 6.5)-methanol (70:30, v/v) in isocratic mode. At a flow-rate of 1.3 ml/min a column pressure of 180–220 bar resulted. Porfiromycin served as internal standard. UV detection was performed at 365 nm. Quantitation limit based on a coefficient of variation <10% in intra- and inter-day assay was 5 μg/l mitomycin C, detection limit based on a signal-to-noise ratio of 3 was 1 μg/l. Recovery was 100% and linearity was shown for the whole range of concentration (1–500 μg/l). None of the five drugs used during chemoembolisation interfered with the assay in vitro. The assay meets the requirements for pharmacokinetic studies of mitomycin C in patients as regards sensitivity and ease of use.     

Determination of formaldehyde in blood plasma by high-performance liquid chromatography with fluorescence detection

An HPLC method was developed for the determination of formaldehyde in human blood plasma. The method was based on the determination of the fluorescent product of the chemical reaction between formaldehyde and ampicillin. A 0.2-ml aliquot of blood plasma was reacted directly with ampicillin under acidic and heating conditions. The reaction product was extracted from the matrix with diethyl ether and analyzed by reversed-phase HPLC with fluorescence detection. Recoveries of spiked formaldehyde at the low ppm (μg/ml) level were between 93% and 102% with relative standard deviations less than 8%. The limits of detection and quantitation of formaldehyde in blood plasma samples were 0.46 μg/ml and 0.87 μg/ml, respectively.     

Determination of pyrazinamide and its main metabolites in rat urine by high-performance liquid chromatography

A new high-performance liquid chromatograhic procedure for simultaneous determination of pyrazinamide (PZA) and its three metabolites 5-hydroxypyrazinamide (5-OH-PZA), pyrazinoic acid (PA), and 5-hydroxypyrazinoic acid (5-OH-PA), in rat urine was developed. 5-OH-PZA and 5-OH-PA standards were obtained by enzymatic synthesis (xanthine oxidase) and checked by HPLC and GC–MS. Chromatographic separation was achieved in 0.01 M KH₂PO₄ (pH 5.2), circulating at 0.9 ml/min, on a C₁₈ silica column, at 22°C. The limits of detection were 300 μg/l for PZA, 125 μg/l for PA, 90 μg/l for 5-OH-PZA and 70 μg/l for 5-OH-PA. Good linearity (r²>0.99) was observed within the calibration ranges studied: 0.375–7.50 mg/l for PZA, 0.416–3.33 mg/l for PA, 0.830–6.64 mg/l for 5-OH-PZA and 2.83–22.6 mg/l for 5-OHPA. Accuracy was always lower than ±10.8%. Precision was in the range 0.33–5.7%. The method will constitute a useful tool for studies on the influence of drug interactions in tuberculosis treatment.     

Simultaneous determination of citalopram, fluoxetine, paroxetine and their metabolites in plasma and whole blood by high-performance liquid chromatography with ultraviolet and fluorescence detection

A method for the simultaneous determination of the three selective serotonin reuptake inhibitors (SSRIs) citalopram, fluoxetine, paroxetine and their metabolites in whole blood and plasma was developed. Sample clean-up and separation were achieved using a solid-phase extraction method with C₈ non-endcapped columns followed by reversed-phase high-performance liquid chromatography with fluorescence and ultraviolet detection. The robustness of the solid-phase extraction method was tested for citalopram, fluoxetine, paroxetine, Cl-citalopram and the internal standard, protriptyline, using a fractional factorial design with nine factors at two levels. The fractional factorial design showed two significant effects for paroxetine in whole blood. The robustness testing for citalopram, fluoxetine, Cl-citalopram and the internal standard revealed no significant main effects in whole blood and plasma. The optimization and the robustness of the high-performance liquid chromatographic separation were investigated with regard to pH and relative amount of acetonitrile in the mobile phase by a central composite design circumscribed. No alteration in the elution order and no significant change in resolution for a deviation of ±1% acetonitrile and ±0.3 pH units from the specified conditions were observed. The method was validated for the concentration range 0.050–5.0 μmol/l with fluorescence detection and 0.12–5.0 μmol/l with ultraviolet detection. The limits of quantitation were 0.025 μmol/l for citalopram and paroxetine, 0.050 μmol/l for desmethyl citalopram, di-desmethyl citalopram and citalopram-N-oxide, 0.12 μmol/l for the paroxetine metabolites by fluorescence detection, and 0.10 μmol/l for fluoxetine and norfluoxetine by ultraviolet detection. Relative standard deviations for the within-day and between-day precision were in the ranges 1.4–10.6% and 3.1–20.3%, respectively. Recoveries were in the 63–114% range for citalopram, fluoxetine and paroxetine, and in the 38–95% range for the metabolites. The method has been used for the analysis of whole blood and plasma samples from SSRI-exposed patients and forensic cases.     

Monitoring of olanzapine in serum by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry

A selective assay of olanzapine with liquid chromatography atmospheric pressure chemical ionization (LC–APCI–MS, positive ions) is described. The drug and internal standard (ethyl derivative of olanzapine) were isolated from serum using a solid-phase extraction procedure (C₁₈ cartridges). The separation was performed on ODS column in acetonitrile–50 mM ammonium formate buffer, pH 3.0 (25:75). After analysis of mass spectra taken in full scan mode, a selected-ion monitoring detection (SIM) was applied with the following ions: m/z 313 and 256 for olanzapine and m/z 327 and 270 for the internal standard for quantitation. The limit of quantitation was 1 μg/l, the absolute recovery was above 80% at concentration level of 10 to 100 μg/l. The method tested linear in the range from 1 to 1000 μg/l and was applied for therapeutic monitoring of olanzapine in the serum of patients receiving (Zyprexa™) and in one case of olanzapine overdose. Olanzapine in frozen serum samples and in frozen extracts was stable over at least four weeks. The examinations of urine extracts from patients receiving olanzapine revealed peaks of postulated metabolites (glucuronide and N-desmethylolanzapine).     

Determination of dichloroanilines in human urine by GC–MS, GC–MS–MS, and GC–ECD as markers of low-level pesticide exposure

Methods for the determination of 3,4-dichloroaniline (3,4-DCA) and 3,5-dichloroaniline (3,5-DCA) as common markers of eight non-persistent pesticides in human urine are presented. 3,5-DCA is a marker for the exposure to the fungicides vinclozolin, procymidone, iprodione, and chlozolinate. Furthermore the herbicides diuron, linuron, neburon, and propanil are covered using their common marker 3,4-DCA. The urine samples were treated by basic hydrolysis to degrade all pesticides, metabolites, and their conjugates containing the intact moieties completely to the corresponding dichloroanilines. After addition of the internal standard 4-chloro-2-methylaniline, simultaneous steam distillation extraction (SDE) followed by liquid–liquid extraction (LLE) was carried out to produce, concentrate and purify the dichloroaniline moieties. Gas chromatography (GC) with mass spectrometric (MS) and tandem mass spectrometric (MS–MS) detection and also detection with an electron-capture detector (ECD) after derivatisation with heptafluorobutyric anhydride (HFBA) were employed for separation, detection, and identification. Limit of detection of the GC–MS–MS and the GC–ECD methods was 0.03 and 0.05 μg/l, respectively. Absolute recoveries obtained from a urine sample spiked with the internal standard, 3,5-, and 3,4-DCA, ranged from 93 to 103% with 9–18% coefficient of variation. The three detection techniques were compared concerning their performance, expenditure and suitability for their application in human biomonitoring studies. The described procedure has been successfully applied for the determination of 3,4- and 3,5-DCA in the urine of non-occupationally exposed volunteers. The 3,4-DCA levels in these urine samples ranged between 0.13 and 0.34 μg/g creatinine or 0.11 and 0.56 μg/l, while those for 3,5-DCA were between 0.39 and 3.33 μg/g creatinine or 0.17 and 1.17 μg/l.     

Simple high-performance liquid chromatography determination of ampicillin in human serum using solid-phase extraction disk cartridges

A simple and reproducible method for the analysis of ampicillin in human serum was developed. Serum samples were extracted using solid-phase extraction disk cartridges containing a sorbent of styrene divinyl/benzene. Extracts were separated by reversed-phase C₁₈ high-performance liquid chromatography with UV detection at 220 nm. The mobile phase consisted of acetonitrile–10 mM NaH₂PO₄ (6.5:93.5, v/v). Using this extraction procedure, recovery from serum was 98.4±5.6%. The quantitation limit was 0.19 μg/ml using 0.5 ml of serum. The calibration curves from 0.19 to 9.41 μg/ml were linear with correlation coefficients of 0.999. This method is suitable for therapeutic drug monitoring of ampicillin (ABPC) after oral administration of lenampicillin hydrochloride.     

Determination of nelfinavir, a potent HIV protease inhibitor, and its active metabolite M8 in human plasma by high-performance liquid chromatography with photodiode-array detection

We developed and characterized a high-performance liquid chromatographic assay for the determination of nelfinavir (NFV), a potent HIV protease inhibitor, and its active metabolite M8 in human plasma. Extraction of the internal standard, M8 and NFV from the plasma buffered at pH 9.5 was achieved by a liquid–liquid extraction with a mixture of methyl-tert.-butyl ether and hexane. Following two washes of the reconstituted sample with hexane, separation was achieved on an octadecylsilyl analytical column with a mobile phase containing 0.1% trifluoroacetic acid–acetonitrile–methanol (51:46:5, v/v). Detection was performed using an ultraviolet photodiode-array detector. The signal was monitored at a wavelength of 220 nm. The assay was found to be linear and has been validated over the concentration range of 25 to 3000 μg/l for M8 and 25 to 6000 μg/l for NFV, from 500 μl of plasma. Recoveries were 98.9% (SD 8.9%), and 100.2% (SD 11.7%) for M8 and NFV, respectively. Concentrations that gave a signal-to-noise ratio of three (15 μg/l for both M8 and NFV) were selected to determine the limit of detection. The lower limit of quantification (25 μg/l for both M8 and NFV) was defined as the concentration for which the relative standard deviation and the percent deviation from the nominal concentration were lower than 20%.     

Development and validation of a high-performance liquid chromatographic method for the determination of methocarbamol in human plasma

An isocratic HPLC method was developed and validated for the quantitation of methocarbamol in human plasma. Methocarbamol and internal standard in 200 μl of human plasma were extracted with ethyl acetate, evaporated to dryness and reconstituted in water. Separation was achieved on a reversed-phase C₁₈ column with a mobile phase of methanol—0.1 M potassium phosphate monobasic—water (35:10:55, v/v/v). The detection was by ultraviolet at 272 nm. Linearity was established at 1–100 μg/ml (r > 0.999). The limit of quantitation was designed as 1 μg/ml to suit pharmacokinetic studies. Inter-day precision and accuracy of the calibration standards were 1.0 to 3.6% coefficients of variance (C.V.) and −2.0 to +1.6% relative error (R.E.). Quality controls of 3, 20 and 70 μg/ml showed inter-day precision and accuracy of 2.5 to 3.6% C.V. and −0.9 to −0.4% R.E. Recovery of methocarbamol was 91.4–100.3% in five different lots of plasma. The method was shown to be applicable on different brands of C₁₈ columns.     

Determination of flumequine and 7-hydroxyflumequine in plasma of sheep by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of flumequine and its metabolite 7-hydroxyflumequine in sheep plasma was described. The two compounds were extracted from 100 μl of plasma by liquid–liquid extraction. Aliquots (100 μl) were injected onto the HPLC system and separated on a LiChrospher Select B column with an isocratic system. The compounds were detected by fluorimetric detection for concentrations below 500 μg/l and by UV detection for the concentrations exceeding 500 μg/l. The range of the validated concentrations were 50 000 to 5 μg/l and 500 to 10 μg/l with mean recovery rates of 87±3% and 60±1% for flumequine and 7-hydroxyflumequine, respectively.     

Comparative In Vitro Activities of Lysostaphin and Other Antistaphylococcal Antibiotics on Clinical Isolates of Staphylococcus aureus

The in vitro activity of lysostaphin against clinical isolates of Staphylococcus aureus was determined by conventional tube-dilution methods. For comparison, minimal inhibitory concentration (MIC) values were also determined for penicillin G, ampicillin, methicillin, ristocetin, vancomycin, and erythromycin. Phage type and penicillinase and coagulase production were determined for each isolate. The MIC values for lysostaphin ranged from <0.047 to 12.5 μg/ml; 96% of the penicillinase-positive strains were inhibited by 1.56 μg/ml of lysostaphin, whereas 3.12 μg/ml of vancomycin and methicillin were required to attain the same degree of inhibition.     

Quantification of 3,4-methylenedioxymetamphetamine and its metabolites in plasma and urine by gas chromatography with nitrogen–phosphorus detection

A gas chromatographic method with nitrogen–phosphorus detection involving a solid–liquid extraction phase was developed and validated for the simultaneous quantification of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) in plasma. A modification of this method was validated for the analysis of MDMA, MDA, 4-hydroxy-3-methoxymethamphetamine (HMMA) and, 4-hydroxy-3-methoxyamphetamine (HMA) in urine. Under the analytical conditions described, the limits of detection in plasma and urine were less than 1.6 μg/l and 47 μg/l, respectively, for all the compounds studied. Good linearity was observed in the concentration range evaluated in plasma (5–400 μg/l) and urine (100–2000 μg/l) for all compounds tested. The recoveries obtained from plasma were 85.1% and 91.6% for MDMA and MDA, respectively. Urine recoveries were higher than 90% for MDMA and MDA, 74% for HMMA, and 64% for HMA. Methods have been successfully used in the assessment of plasma and urine concentrations of MDMA and its main metabolites in samples from clinical studies in healthy volunteers.     

High-performance liquid chromatographic assay of bromocriptine in plasma and eye tissue of the rabbit

A reliable reversed-phase high-performance liquid chromatographic method has been developed for the determination of bromocriptine (BCT) in plasma and eye tissues. The BCT and propranolol, added as an internal standard (I.S.), were extracted by a liquid–liquid technique followed by an aqueous back-extraction, allowing injection of an aqueous solvent into a 4-μm Nova-Pak C₁₈ column (150×3.9 mm I.D.). The mobile phase was a mixture of 30 parts of acetonitrile and 70 parts of 0.2% triethylamine (pH 3) at a flow-rate of 1 ml/min. Fluorescence detection was at an excitation wavelength of 330 nm and an emission wavelength of 405 nm. The retention times of I.S. and BCT were 4.1 and 11.6 min, respectively. The calibration curve was linear over the concentration range 0.2–10 μg/l for plasma (r>0.999) and vitreous humour (r>0.997) and 1–50 μg/l for aqueous humour (r>0.985). The limit of quantification was 0.2 μg/l for plasma and vitreous humour using a 1-ml sample and was 1 μg/l for aqueous humour using a 0.2-ml sample. The quality control samples were reproducible with acceptable accuracy and precision. The within-day recovery (n=3) was 100–102% for plasma, 91–106% for aqueous humour and 96–111% for vitreous humour. The between-day recovery (n=9) was 90–114% for plasma, 83–115% for aqueous humour and 90–105% for vitreous humour. The within-day precision (n=3) and the between-day precision (n=9) were 1.7–7.0% and 8.1–13.6%, respectively. No interferences from endogenous substances were observed. Taken together, the above simple, sensitive and reproducible high-performance liquid chromatography assay method was suitable for the determination of BCT in plasma and eye tissues following ocular application of BCT for the therapy of myopia.     

Simultaneous high-performance liquid chromatographic assay of droperidol and flunitrazepam in human plasma: Application to haemodilution blood samples collected during clinical anaesthesia

A simultaneous assay for droperidol and flunitrazepam by high-performance liquid chromatography has been developed and applied to blood samples collected during an acute normovolemic haemodilution under general anaesthesia. Haemodilution blood samples were stored at +4°C to be transfused, if required, to a patient during the post-surgical phase. A C₁₈ Supelclean cartridge was used for solid-phase extraction, and the recoveries were 74% and 89%, respectively, for droperidol and flunitrazepam. Compounds were chromatographed on a C₁₈ Novapak column at 250 nm, with a mobile phase of acetonitrile—10 mM ammonium acetate buffer (pH 6.7) (45:55, v/v). Nitrazepam was used as the internal standard. For both drugs, the assay was linear up to 500 μg/l, and the detection limits were 20 and 10 μg/l for droperidol and flunitrazepam, respectively, and their observed levels in haemodilution samples were 93 ± 82 μg/l and 76 ± 107 μg/l, respectively. Some of the values for flunitrazepam were higher than the minimal efficient concentration, defined as the plasma level observed at the time of the patient wakening from anaesthesia (12 ± 4 μg/l). According to our results, haemodilution sampling can be performed before induction of anaesthesia. When the blood is collected after the anaesthetic induction, it seems necessary to determine levels of the two drugs in haemodilution samples to avoid side-effects.     

Fast liquid chromatographic determination of urinary trans,trans-muconic acid

trans,trans-Muconic acid (1,3-butadiene-1,4-dicarboxylic acid, MA), a minor urinary metabolite of benzene exposure, was determined, after clean-up by solid-phase anion-exchange chromatography, by reversed-phase HPLC on a C₁₈ column (5 × 0.46 cm I.D., 3 μm particle size), using formic acid-tetrahydrofuran-water (14:17:969) as mobile phase and UV detection at 263 nm. The recovery of MA from spiked urine was > 95% in the 50–500 μg/l range; the quantification limit was 6 μg/l; day-to-day precision, at 300 μg/l, C.V. = 9.2%; the run time was less than 10 min. Urinary MA excretion was measured in two spot urine samples of 131 benzene environmentally exposed subjects: midday values obtained in non-smokers (mean±S.D.=77±54 μg/l, N = 82) were statistically different from those of smoerks (169±85 μg/l, N = 30) (P<0.0001); each group showed a statistically significant increase between MA excretion in midday over morning samples. Moreover, in subjects grouped according to tobacco-smoke exposure level, median values of MA were positively associated with and increased with daily smoking habits.