Determination of Penicillinase-Resistant Penicillins In Serum Using High-Pressure Liquid Chromatography

Abstract

A method for the determination of methicillin, oxacillin, cloxacillin, dicloxacillin, and nafcillin in serum using high-pressure liquid chromatography (HPLC) is described. The drugs were extracted from serum using a two-step procedure employing acetonitrile followed by methylene chloride. The extraction procedure concentrated the antibiotics in a smaller volume which allows more accurate determinations of low serum levels. The treated sera were analyzed by HPLC on a reverse-phase column and detected by ultraviolet light absorption at 254 nm. Serum concentrations were measurable as low as 0.5 μg/ml. Recovery procedures showed less than 2.5% variation in peak heights when the antibiotics were extracted from different pools of serum. No interfering absorption was found in extracts of serum samples pooled from healthy volunteers, from a commercial source, or from two serum pools from patients receiving a variety of other drugs. Two spiked serum specimens prepared for each antibiotic were assayed four times by HPLC and by the microbiological agar diffusion method. No significant statistical differences between the methods were observed. Control materials were assayed for between-batch and within-batch reproducibility in the presence or absence of an internal standard. Results for between-batch reproducibility demonstrate CV's of about 5%. This procedure provides a sensitive, specific, accurate, and rapid method for determining antibiotic levels in routine clinical specimens.     

Microwave-assisted extraction of tricyclic antidepressants from human serum followed by high performance liquid chromatography determination

A microwave-assisted extraction (MAE) method has been developed and optimized for the extraction of six tricyclic antidepressants (TCAD; nordoxepin, nortriptyline, imipramine, amitriptyline, doxepin, dezypramine) from human serum. Optimal parameters of MAE (solvent and extraction temperature) for water solution of these drugs were defined. The microwave-assisted procedure developed was validated by extraction of serum samples at two concentration levels and then successfully applied to the analysis of reference material. Limit of quantification, precision and recovery were found for the studied compounds in the ranges 0.04-0.15 microg/mL, 1.57-4.34% (RSD) and 94-105%, respectively.     

Development and validation of a reference measurement procedure for certification of phenytoin, phenobarbital, lamotrigine, and topiramate in human serum using isotope-dilution liquid chromatography/tandem mass spectrometry

Phenytoin (PHT), phenobarbital (PHB), lamotrigine (LTG), and topiramate (TPM) are some of the most widely used antiepileptic drugs (AEDs). Monitoring of their concentrations in serum is important for the treatment of epilepsy. A reference measurement procedure (RMP) for certification of PHT, PHB, LTG, and TPM in serum has been developed and critically evaluated. Isotopically labeled compounds of PHT, PHB, LTG, and TPM are used as internal standards for the four AEDs. The four drugs and their respective labeled internal standards are simultaneously extracted from serum using solid-phase extraction prior to reversed-phase liquid chromatography–tandem mass spectrometry (LC-MS/MS). Chromatographic separation was performed using a C₁₈ column. Electrospray ionization (ESI) in the positive ion mode for PHT and LTG, and in the negative ion mode for PHB and TPM were used. The recovery of AEDs added to serum (accuracy of the extraction method) was evaluated by recovery studies of measuring the four drugs in spiked samples with known drug levels. The recoveries of the added drugs ranged from 98.6% to 102.0%. The absolute recoveries (extraction efficiencies) of the four drugs with this method ranged from 97% to 100%. Excellent repeatability was obtained for the four drugs with between-set coefficients of variation (CVs) within 1%. The type B components estimates are conservatively large and are considerably larger than the type A component. Therefore, we use the usual metrological expansion factor of 2 to provide an approximate 95% coverage interval. The relative expanded uncertainties for the four AEDs ranged from 2.3% to 2.4%. This LC-MS/MS RMP for PHT, PHB, LTG, and TPM in serum demonstrating good accuracy and precision can be used to assess the accuracy of routine methods used in clinical laboratories.     

Determination of pesticides in apples by liquid chromatography with electrospray ionization tandem mass spectrometry and estimation of measurement uncertainty

A liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated to determine 90 pesticides in apple samples. MS/MS data acquisition was achieved by applying multiple-reaction monitoring of 2 fragment ion transitions to provide a high degree of sensitivity and selectivity for both quantitation and confirmation. Matrix-matched standard calibration curves with the use of isotopically labeled standards (or a chemical analog) as internal standards were used to achieve the best accuracy for the method. Both a conventional method validation procedure and a designed experiment were applied to study the accuracy and precision of the method. A compiled computer program that provided a semiautomated procedure for handling a large number of calculations was used to calculate the overall recovery, intermediate precision, and measurement uncertainty (MU). In general, the overall recoveries from samples spiked at levels of 10, 50, and 80 microg/kg, ranged from 60.8 to 121.1%, intermediate precision was <10%, and MUs were <30%. Poor accuracy and/or repeatability was observed for pyridate and etofenprox. The method limits of detection based on a signal-to-noise ratio of >3 were usually <1 microg/kg (ppb), except those for aldicarb sulfoxide and pyridaphenthion, which were about 5 microg/kg.     

Application of microextraction by packed sorbent to isolation of psychotropic drugs from human serum

A method of microextraction by packed sorbent (MEPS) followed by liquid chromatography with diode array detection has been developed and optimized for the extraction of six tricyclic antidepressants (amitriptyline, nortriptyline, imipramine, desipramine, doxepin, nordoxepin) from human serum. The optimal parameters of MEPS extraction (type of sorbent, volume of sample, composition, and volume of washing and elution solutions) for these drugs in spiked samples were defined. The developed MEPS procedure was validated and then successfully applied to the analysis of serum reference material. The limit of detection (0.02–0.05 μg/mL), intraday (2.7–8.8%) and interday (4.4–11.6%) precision (RSD), and the accuracy of the assay (94.5–108.8%) at three concentration levels—0.2, 0.5, and 0.8 μg/mL—were estimated. The accuracy of the method was evaluated by the analysis of certified reference material. Moreover, the validated procedure was compared with the solid-phase extraction technique. Finally, microextraction by packed sorbent was assessed as a suitable tool in forensic and clinical methods for serum sample preparations.     

Clozapine imprinted polymers: synthesis, characterization and application for drug assay in human serum

A variety of molecularly imprinted polymers (MIPs) against clozapine (CLZ) were synthesized and their recognition properties were compared with blank non-imprinted polymers. Methacrylic acid (MAA) was used as a functional monomer and Chloroform or tetrahydrofuran (THF) were applied as polymerization solvents. Chloroform as the solvent and MAA/CLZ ratio of 5 was selected as optimized polymerization condition. In Scatchard analysis of MIP-CLZ interactions, two classes of binding sites were found in MIP—high affinity (KD = 14.5 μM) and low affinity (KD = 322.5 μM) binding sites. The polymer was evaluated as a selective sorbent in molecularly imprinted solid-phase extraction (MISPE) of CLZ from human serum. The MISPE procedure was developed and optimized with a recovery of 88-102%. The intra- and inter-day precision values were less than 1.36% and 2.5%, respectively. The selectivity of MISPE for CLZ was studied in comparison with some drugs. These drugs could be present with CLZ, simultaneously in serum of patients. The data indicated that the imprinted polymer had a good selectivity and affinity for CLZ and could be used for selective extraction and analysis of CLZ in human serum.     

Development and evaluation of a candidate reference measurement procedure for the determination of testosterone in human serum using isotope dilution liquid chromatography/tandem mass spectrometry

A candidate reference measurement procedure for total testosterone in human serum involving isotope dilution (ID) coupled with liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed and critically evaluated. The endogenous testosterone and its internal standard (testosterone-d ₃) were extracted from the serum matrix using a combination of solid-phase extraction and liquid–liquid extraction prior to reversed-phase LC/MS/MS. Accuracy of the measurements was evaluated by a recovery study using testosterone-spiked serum. The recovery of the added testosterone ranged from 100.0 to 100.3%. This method was applied to the determination of testosterone in frozen serum samples from three individual donors (one female and two males) with the testosterone concentrations ranging from 0.3 to 8.5 ng g⁻¹. Repeatability with within-set coefficients of variation (CVs) from 0.1 to 1.0% and intermediate precision with between-set CVs from 0.1 to 0.5% for both female and male serum materials were demonstrated. Excellent linearity was obtained for all linear regression lines. The detection limit at a signal-to-noise ratio of approximately 3 was 2 pg of testosterone in serum. Structural analogs as well as testosterone metabolites were tested and found to not interfere with the measurement of testosterone. This well-characterized LC/MS/MS method for serum testosterone, which demonstrates good accuracy and precision, and low susceptibility to interferences, qualifies as a reference measurement procedure that can be used to provide an accuracy base to which routine methods for testosterone can be compared and that will serve as a standard of higher order for measurement traceability.     

Determination of antimigraine compounds rizatriptan, zolmitriptan, naratriptan and sumatriptan in human serum by liquid chromatography/electrospray tandem mass spectrometry

Development of a rapid, sensitive and selective method for the determination of antimigraine drugs from human serum is essential for understanding the pharmacokinetics of these drugs when administered concurrently. Solid phase extraction (SPE) using Oasis HLB was used to extract the drugs (sumatriptan, naratriptan, zolmitriptan and rizatriptan) and the internal standard bufotenine from serum. A method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated to simultaneously quantitate these antimigraine drugs from human serum. The precursor and major product ions of the analytes were monitored on a triple quadrupole mass spectrometer with positive ion electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The base peak in all the analytes is formed by alpha cleavage associated with protonation of the secondary amine. Mechanisms for the formation of the collision-induced dissociation products of these antimigraine compounds are proposed. Linear calibration curves were generated from 1-100 ng/mL with all coefficients of determination greater than 0.99. The inter- and intraday precision (%RSD) were less than 9.3% and accuracy (%error) was less than 9.8% for all components. The limits of detection (LOD) for the method were 250 pg/mL for sumatriptan and 100 pg/mL for the remaining analytes based on a signal-to-noise ratio of 3.     

Determination of tebuconazole, trifloxystrobin and its metabolite in fruit and vegetables by a Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method using gas chromatography with a nitrogen-phosphorus detector and ion trap mass spectrometry

A new analytical method using QuEChERS procedure by gas chromatography with a nitrogen-phosphorus detector (GC-NPD) and ion trap mass spectrometry (GC-IT-MS) for the quantitative determination of tebuconazole, trifloxystrobin and its metabolite trifloxystrobin acid has been developed and validated. The analytes were extracted from five fruit and vegetable matrices using acetonitrile and subsequently cleaned up using primary secondary amine (PSA) or octadecylsilane (C18) as sorbent prior to GC analysis. The present methods provided sufficient sensitivity as reflected by the values of limit of detection (LOD) and limit of quantification (LOQ) of 0.4-7 and 1.2-20 μg/kg for GC-IT-MS/MS and GC-NPD. The recoveries were, on average, 68-117 and 68-121%, respectively, for three compounds by GC-NPD and GC-IT-MS/MS with intra-day precision achieved with an RSD of 2.7-19.1%. The inter-day precision was better than 15.1% as determined by GC-NPD. The QuEChERS procedure, by using two sorbents (PSA and C18) and the matrix-matched standards, gave satisfactory recoveries and RSD values in different matrices. IT-MS acquisition provided higher specificity and selectivity for pesticides and better limit of detection and quantification. However, the repeatability and precision of NPD method were better compared with IT-MS.     

Rapid and simple determination of selenium in blood serum by inductively coupled plasma–mass spectrometry (ICP–MS)

An inductively coupled plasma mass spectrometer (ICP-MS) with a rapid sample-preparative procedure was used for the determination of selenium in blood serum. Blood serum was prepared by dilution in an acidic solution consisting of nitric acid (1%), X-triton (0.1%) and 1-butanol (0.8%). A calibration curve was established for 1-40 microg mL(-1) (r(2)>0.99). The limit of detection was 0.5 microg mL(-1). Repeatability and intermediate precision were satisfactory with relative standard deviations (RSD) of 2.0% and 3.2%, respectively. This method was easily applied to reference materials with satisfactory accuracy. Good correlation (r(2)=0.96) was observed between ICP-MS and atomic absorption spectrometry (AAS) for the determination of (82)Se in blood serum from 23 patients. These results suggest that the sample preparative procedure coupled with ICP-MS can be used for the routine determination of (82)Se in human blood serum.     

Direct injection determination of benzoylecgonine, heroin, 6-monoacetylmorphine and morphine in serum by MLC

A simple and sensitive direct injection chromatographic procedure is developed for the determination of heroin, two of its metabolites (morphine and 6-monoacetylmorphine (6-MAM)), and benzoylecgonine (a metabolite of cocaine) in serum samples. The proper resolution of the four substances is obtained with a chemometrics approach, where the retention is modelled as a first step using the retention factors obtained in a limited number of mobile phases. Afterwards, an optimisation criterion that takes into account the position and shape of the chromatographic peaks is applied. The mobile phase selected to carry out the analysis was 0.1 mol L(-1) SDS-4% (v/v) butanol buffered at pH 7, in which the separation is performed in less than 18 min. The limits of quantification were in the 17-36 ng mL(-1) range. Intra- and inter-day assay accuracy and precision (below 3%) were obtained following ICH guidelines. The method developed was applied to the determination of the drugs studied in serum samples with good recoveries (90-104%). Serum samples from subjects that have been ingested cocaine and heroin were also analysed. The samples were injected directly in the chromatographic system without any pretreatment.     

Quantification of retinoid concentrations in human serum and brain tumor tissues

Retinoic acid signaling is essential for central nervous system (CNS) differentiation and appears to be impaired in tumors. Thus far, there are no established methods to quantify relevant retinoids (all-trans-retinoic acid, 9-cis-retinoic acid, 13-cis retinoic acid, and retinol) in human brain tumors. We developed a single step extraction and quantification procedure for polar and apolar retinoids in normal tissue, lipid-rich brain tumor tissues, and serum. This quantification procedure is based on high performance liquid chromatography (HPLC) with diode-array detection (DAD) using all-trans-acitretin as an internal standard and extraction by liquid–liquid partition with ethyl acetate and borate buffer at pH 9. Recovery with this extraction procedure was higher than earlier (two-step) liquid–liquid extraction procedures based on hexane, NaOH, and HCl. The overall quantification procedure was validated according to Food and Drug Administration (FDA) guidelines and fulfilled all criteria of accuracy, precision, selectivity, recovery, and stability. The overall method accuracy varied between −5.6% and +5.4% for serum and −3.8% and +6.2% for tissues, and overall precision ranged from 3.1% to 6.9% for serum and 2.1% to 8.3% for tissues (%CV batch-to-batch). The lower limit of quantification for all compounds in tumor tissue (and serum) was 3.9 ng g⁻¹ (ng mL⁻¹). Using this assay, photodegradation of the retinoids was evaluated and endogenous polar and apolar retinoids were quantified in sera and brain tumor tissues of patients and compared with serum and tonsil tissue concentrations of controls. It may thus serve as a suitable method for the characterization of retinoid uptake and metabolism in the respective compartments.     

Photo- and thermal-stability studies on benzimidazole anthelmintics by HPLC and GC-MS

Photo- and thermal-stability of the anthelmintics Albendazole, Mebendazole and Fenbendazole as in solid as in solution form has been investigated, by using a Xenon arc lamp as a radiation source, according to the ICH guideline for the drug stability tests. The degradation process was monitored by a HPLC method. All drugs showed high photosensitivity in solution but a reliable stability in solid form and when exposed to a temperature up to 50 degrees C. Two main degradation products from hydrolysis of the carbamic groups were identified by GC-MS. Validation studies demonstrated high accuracy (recovery 94 to 106%) and precision (RSD under 4.6%) of the HPLC method. The analytical procedure was successfully applied to the control of the drugs in the respective pharmaceutical formulations.     

高效液相色谱法同时测定血清和尿中厚朴酚与和厚朴酚

 建立了大鼠服用厚朴提取物后的血清中及尿中厚朴酚与和厚朴酚的高效液相色谱测定法。色谱柱填料为SpherisorbC18,流动相为甲醇-水-冰醋酸(体积比为70∶30∶1),UV检测波长为294nm,灵敏度0.005AUFS。样品用甲醇沉淀蛋白,上清液酸化后用乙酸乙酯-乙醚萃取,然后测定其中的药物浓度。血清和尿中的药物浓度与峰面积的线性关系良好,线性范围分别为0.05～2mg/L(厚朴酚)、0.025～1mg/L(和厚朴酚);精密度和重现性良好。血清中厚朴酚与和厚朴酚的平均加样回收率分别为95.6%(RSD=3.85%)和93.8%(RSD=3.95%),尿中分别为96.0%(RSD=3.83%)和94.9%(RSD=3.54%)。     

Separation and determination of selected psychotropic drugs in human serum by SPE/HPLC/DAD on C18 and Polar-RP columns

A simple and sensitive high-performance liquid chromatographic method with diode array detection is described for the quantification of some psychotropic drugs: fluoxetine, sertraline, alprazolam, perphenazine, zolpidem, and hydroxyzine in fortified human serum samples. The test compounds were extracted from human serum by solid-phase extraction using C18 extraction column and injected into C18 or Polar-RP analytical columns for separation. A mobile phase was composed of methanol, water, acetate buffer at pH 3.5, and diethylamine. The method was validated for the concentration range 0.4–5?µg?mL^?1 for zolpidem and 0.5–6?µg?mL^?1 for other drugs. Mean recoveries were from 87.79% to 107.94% with adequate precision (% RSD ≤2.1%). The full separation of all investigated drugs, good peaks’ symmetry, and simultaneously high system efficiency were obtained on Polar-RP column, which was used for the first time to analyze these drugs. System efficiency obtained on the column was significantly higher compared to that obtained on commonly used C18 column. The method seems to be suitable for the analysis of investigated drugs in human plasma for psychiatric patients in multiple drug overdoses as well as for control of pharmacotherapy, particularly in combination therapy.     

Determination of trapidil in human serum and urine by derivative UV spectrophotometry after selective solid-phase extraction

A novel analytical technique able to determine the anti-ischemic drug trapidil in human serum and urine is proposed. In order to achieve satisfactory sensitivity and selectivity, an extraction procedure was required to isolate the drug from complex matrixes such as serum and urine. A solid-phase extraction procedure was investigated to both increase the analyte concentration and eliminate the interfering molecules present in large amounts in both matrixes. Optimization of the extraction step was realized by selecting a new polymeric sorbent based on a surface-modified styrene–divinylbenzene polymer which provided fast and efficient drug extraction. Drug quantification was performed by using the third-order derivative spectra of the SPE eluates. Absorbance specific signals at ³D_335,316 and ³D₃₁₆ nm for urine and serum, respectively, were demonstrated to be directly proportional to drug concentration and barely affected by residual matrix interferences. Under the optimized experimental conditions the calibration plots were linear over the concentration range 0.2–50 μg mL⁻¹. The method was validated by analysis of a series of spiked samples. Accuracy (recovery of 95 and 94% for serum and urine, respectively) and precision (RSD below 4%) were good. Figure Assay of Trapidil in biological fluids by SPE and derivative spectrophotometry     

High-performance liquid chromatographic determination of netilmicin in guinea-pig and human serum by fluorodinitrobenzene derivatization with spectrophotometric detection

A high-performance liquid chromatographic procedure for netilmicin determination in guinea-pig and human serum using pre-column derivatization with 1-fluoro-2,4-dinitrobenzene and UV detection is described. Linearity was established over the range 0.5-40 micrograms/ml using only 50 microliters of serum. Accuracy and precision were good, with a mean coefficient of variation less than 5% and a mean relative error less than 4%. This procedure correlates well with an enzyme multiplied immunoassay technique and has a sensitivity similar to those of published fluorescence derivatization methods.     

Determination of salicylate in blood serum by flow injection with immobilized salicylate hydroxylase

A flow injection (FI) enzymatic system, based on the use of immobilized salicylate hydroxylase in glass beads, was developed for the determination of salicylate. Salicylate hydroxylase and nicotinamide adenine dinucleotide (NADH) are used to convert salicylate to catechol. The reaction of catechol with 4-aminophenol at high pH yields a colored product which is detected spectrophotometrically at 565 nm. Ten samples of human serum containing from 5.0 x 10(-4) to 5.0 x 10(-3) mol/L added salicylate were analyzed and the recovery was determined. Eight additional serum samples containing salicylate were analyzed by the Trinder test and the proposed method. The results obtained with the 2 methods showed good agreement by the statistical Student's t-test. The relative precision of the method is about 3.4% (RSD of the mean recovery). Considering the lowest concentration analyzed, the quantitative limit of detection is about 0.2 x 10(-5) mol/L (3 x SD). The volume of the sample used was 150 microL. The proposed method was also used to analyze medicines containing acetylsalicylic acid. The results were statistically compared with those obtained through the U.S. Pharmacopoeia procedure and showed excellent agreement.     

Liquid Chromatographic Analysis of Ciprofloxacin and Ciprofloxacin Metabolites in Body Fluids

Abstract

An isocratic HPLC assay procedure for analysis of ciprofloxacin and three metabolites was developed. The procedure requires only dilution of bile, saliva, and urine samples prior to reverse-phase chromatography on a polystyrene-divinylbenzene (PSDVB) column; analysis of serum samples requires a cleanup step on a PSDVB cartridge prior to chromatography. The dependence of chromatographic efficiency on flow rate and temperature was investigated and the accuracy, precision, selectivity, and sensitivity of the procedure were evaluated. The developed procedure was also compared to a modified version of a published ciprofloxacin procedure that requires an octadecyl-silane (ODS) column for chromatographic separation. Similar efficiency, precision, and accuracy were observed with both procedures and both were used for analysis of clinical samples. However, the procedures were used for different purposes. The PSDVB procedure, because of more favorable column selectivity, was used to assay ciprofloxacin and its metabolites in bile, urine and saliva samples. The ODS procedure, because of a simpler serum preparation step, was used t o assay ciprofloxacin in serum samples.     

A Capillary GC Method Using Nitrogen Phosphorus Detection for Determination of Topiramate in Patients with Epilepsy

A new capillary gas chromatographic assay with nitrogen phosphorus detection for the determination of topiramate in the serum was developed and validated. The sample procedure included a liquid–liquid extraction of 0.1 mL of alkalized sample with ethyl acetate. The organic solvent was evaporated, and flash methylation of topiramate and internal standard 5-(p-methylphenyl)-5-phenylhydantoin with trimethylanilinium hydroxide (0.07 mol L⁻¹ in methanol) was performed. The structure of derivatization product was confirmed using gas chromatography–mass spectrometry. Validation experiments certified suitability of bioanalytical method in the monitoring of serum concentrations in epileptic patients. The limit of detection was found to be 1.69 μmol L⁻¹. The range of applicability and linearity was established from 4.35 to 69.62 μmol L⁻¹. The accuracy and precision reached acceptable values from 86.15 to 109.5% (recovery) respectively, values were from 2.19 to 11.03% (RSD). No interference was found from endogenous substances or studied antiepileptic drugs.