Therapeutic drug monitoring of flecainide in serum using high-performance liquid chromatography and electrospray mass spectrometry

High-performance liquid chromatography with electrospray mass spectrometry (LC-MS) was used for analysis of the drug flecainide in serum. The clean-up was performed by solid-phase extraction, and an aromatic ring positional isomer was used as internal standard. Results from method validation on spiked serum samples showed excellent reproducibility; intra- and inter-assay variations (C.V.% and %Bias) were less than 6% within the therapeutic concentration range of the drug (0.2-1.0 microg/ml). Linearity was demonstrated from 0.05 to 2.0 microg/ml. The limit of detection and quantification was 0.025 and 0.05 microg/ml, respectively. Due to the high selectivity of the mass spectrometric detection, no interferences were observed. Results from clinical samples (n=18) from patients in treatment with Tambocor (flecainide acetate) showed excellent correlation with parallel data obtained from a method based on high-performance liquid chromatography (HPLC) with fluorescence detection after liquid/liquid extraction. The chromatographic separation of flecainide and internal standard was improved compared to earlier HPLC methods. The methodology is simple, accurate and requires only 0.25 ml of sample. It is a well suited method for routine therapeutic drug monitoring in a hospital or clinical chemistry laboratory.     

Determination of 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid in urine using high-performance liquid chromatography and electrospray ionization mass spectrometry.

High-performance liquid chromatography with electrospray ionization mass spectrometry was used to determine 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in urine. After basic hydrolysis of conjugates, the compound was extracted using SPEC-PLUS-3ML-C18 solid-phase extraction columns. A deuterium labelled internal standard (d3-THC-COOH) was added prior to hydrolysis. Separation was performed on a reversed-phase Zorbax Eclipse XDB-C8 analytical column (150x3.0 mm I.D.) using a gradient program from 60 to 80% acetonitrile (4 mM formic acid) at a flow-rate of 0.5 ml/min. The compounds were detected by single ion monitoring of m/z 345 and m/z 348 for the protonated molecules [THC-COOH+H]+ and [d3-THC-COOH+H]+, respectively. The precision and accuracy were tested on spiked urine samples in the range 2.5-125 ng/ml. The mean recovery was 95% (n = 58), coefficients of variations were 2.2-4.3% and the limit of detection 2 ng/ml. Diagnostic qualifying ions of THC-COOH (m/z 327 and m/z 299) and d3-THC-COOH (m/z 330) were generated using up-front collision-induced dissociation. The relative ion intensities in clinical samples (n = 21) were within +/-20% deviation compared with standards. Using this tolerance and the presence of the ions m/z 327 and m/z 299 at the correct retention times as the acceptance criteria for identification of THC-COOH positive samples, the limit of detection was 15 ng/ml. The LC-MS method complies with the current recommendations on drugs of abuse testing, in which mass spectrometric detection is emphasized.     

Analysis of cocaethylene, benzoylecgonine and cocaine in human urine by high-performance thin-layer chromatography with ultraviolet detection: a comparison with high-performance liquid chromatography

Cocaine and ethanol are frequently used at the same time, resulting in the formation of cocaethylene by transesterification. We studied the capability of high-performance thin-layer chromatography (HPTLC) to simultaneously detect cocaethylene, cocaine and benzoylecgonine in 16 urine specimens of drug addicts, previously tested as positive for benzoylecgonine at immunoenzymatic screening. Accuracy and precision, as well as detection and quantitation limits of the method, were evaluated by comparison with high-performance liquid chromatography (HPLC). HPTLC limit of quantitation was 1.0 microg/ml for the three compounds, whereas HPLC limits were 0.2 microg/ml for benzoylecgonine and cocaine, and 0.1 microg/ml for cocaethylene. The relative standard deviation (RSD) ranged from 1.03 to 12.60% and from 1.56 to 16.6% for intra- and inter-day HPTLC analysis, respectively. In the case of the HPLC method, the RSD for the intra-day precision ranged from 0.79 to 5.05%, whereas it ranged from 1.19 to 10.64% for the inter-day precision. In comparison with HPLC, HPTLC is less expensive and faster, requiring 2-3 h to analyze 10-12 samples on a single plate. In conclusion, HPTLC is suitable for determinations of the three analytes only for samples with high concentrations.     

High-performance liquid chromatographic analysis of AQ4N, an alkylaminoanthraquinone N-oxide

A simple, highly selective and reproducible reversed-phase high-performance liquid chromatography method has been developed for the analysis of the new anti-cancer pro-drug AQ4N. The sample pre-treatment involves a simple protein precipitation protocol, using methanol. Chromatographic separations were performed using a HiChrom HIRPB (25 cmX4.6 mm I.D.) column, with mobile phase of acetonitrile-ammonium formate buffer (0.05 M) (22:78, v/v), with final pH adjusted to 3.6 with formic acid. The flow-rate was maintained at 1.2 ml min(-1). Detection was via photodiode array performed in the UV range at 242 nm and, since the compounds are an intense blue colour, in the visible range at 612 nm. The structurally related compound mitoxantrone was used as internal standard. The validated quantification range of the method was 0.05-10.0 microg ml(-1) in mouse plasma. The inter-day relative standard deviations (RSDs) (n=5) ranged from 18.4% and 12.1% at 0.05 microg ml(-1) to 2.9% and 3.3% at 10.0 microg ml(-1) for AQ4N and AQ4, respectively. The intra-day RSDs for supplemented mouse plasma (n=6) ranged from 8.2% and 14.2% at 0.05 microg ml(-1) to 7.6% and 11.5% at 10.0 microg ml(-1) for AQ4N and AQ4, respectively. The overall recovery of the procedure for AQ4N was 89.4 +/- 1.77% and 76.1 +/- 7.26% for AQ4. The limit of detection was 50 ng ml(-1) with a 100 microl sample volume. The method described provides a suitable technique for the future analysis of low levels of AQ4N and AQ4 in clinical samples.     

Quantitative analysis of fentanyl in rat plasma by gas chromatography with nitrogen-phosphorus detection

A sensitive assay method was developed to determine fentanyl, an opiate agonist, in rat plasma by gas chromatography with nitrogen-phosphorus detection. For the pretreatment of plasma samples, sodium hydroxide was added to denature protein and n-butyl chloride was used to extract fentanyl. The calibration curve was linear within the concentration range 0.5 to 50 ng/ml (r=0.9997). The limit of detection was 0.1 ng/ml, and 0.5 ng/ml could be quantified with acceptable precision. Furthermore, fentanyl could be determined in only 200 microl of rat plasma. The method has been successfully applied to an intramuscular pharmacokinetic study at a dose of 10 microg/kg. Therefore, the current method is a valuable analytical tool for investigating the pharmacokinetics of fentanyl at low clinical doses.     

Cellular chromosome DNA interferes with fluorescence quantitative real-time PCR detection of HBV DNA in culture medium

Fluorescence quantitative real-time PCR (FQ-PCR) is a recently developed technique increasingly used for clinical diagnosis by detection of hepatitis B virus (HBV) DNA in serum. FQ-PCR is also used in scientific research for detection of HBV DNA in cell culture. Understanding potential FQ-PCR interference factors can improve the accuracy of HBV DNA quantification in cell culture medium. HBV positive serum was diluted with culture medium to produce three test groups with HBV DNA levels of 5 x 10(7) copies/ml (high), 5 x 10(5) copies/ml (medium), and 5 x 10(3) copies/ml (low). Chromosome DNA was extracted from HepG2 cells and then added to high, medium, and low group samples at final concentrations of 0, 12.5, 25, 50, and 100 microg/ml. The samples were quantified by FQ-PCR and data were evaluated using statistical software. No marked changes were seen in the quantitative curves for high level HBV DNA samples when the samples were supplemented with 0-100 microg/ml of chromosome DNA. Interference was observed in medium level samples when 50 and 100 microg/ml of chromosome DNA was added. Interference was also observed in low level HBV DNA samples when the concentration of added chromosome DNA was greater than 25 microg/ml. The interference was eliminated when samples were digested by DNase I prior to PCR detection. In Conclusions, the presence of cellular chromosome DNA can interfere with the detection of HBV DNA by FQ-PCR. Removal of cellular chromosome DNA from culture media prior to FQ-PCR is necessary for reliable HBV DNA quantitative detection.     

Rapid high-performance liquid chromatographic determination of serum gabapentin.

Gabapentin (GBP) is a new antiepileptic drug approved for clinical treatment of partial seizures in the USA. Serum GBP concentrations in 283 patients were studied using high-performance liquid chromatography with fluorescence detection. The standard curves were linear over a range of 60 ng to 15 microg/ml. The coefficient of variations were 3.4 to 8.8% and 1.4 to 9.8% for intra- and inter-assay studies, respectively. The lower limit of quantitation was 10 ng/ml. Of the 283 patients studied, 72.5% had GBP levels between 2 and 10 microg/ml, 14.8% were below 2 microg/ml and 12.7% above 10 microg/ml. The mean+/-S.E. of GBP in 283 patients was 5.38+/-0.23 microg/ml. Peak concentrations of more than 15 microg/ml and trough levels as low as 0.1 microg/ml were not uncommon. The method described was rapid, simple, highly sensitive and reproducible. Other antiepileptic drugs and endogenous compounds did not interfere with the assay.     

An in-vivo study on placental transfer of naproxen in early human pregnancy

BACKGROUND: Naproxen is one of the most common non-steroidal anti-inflammatory drugs used by women of reproductive age. Naproxen is known to be teratogenic in animals. The aim of this study was to investigate the placental transfer of naproxen in the first trimester of human pregnancy, and to determine the amount of the drug in different embryonic compartments. METHODS: Twenty-eight patients who requested surgical termination of pregnancy in the first trimester were given two oral 500 mg doses of naproxen before the surgical procedure. Four biological samples, maternal venous blood, coelomic fluid, amniotic fluid and fetal tissue, were collected from each patient for drug analyses by high performance liquid chromatography. RESULTS: Naproxen was detected in all samples. The mean (+/- SD) concentrations were 69.5 +/- 12.2 microg/ml, 6.4 +/- 2.4 microg/g, 1.85 +/- 1.03 microg/ml and 0.14 +/- 0.11 microg/ml in maternal serum, fetal tissue, coelomic fluid and amniotic fluid respectively. The mean amniotic fluid/maternal drug ratio and fetal/maternal drug ratio were 0.002 (range 0.0005-0.0064) and 0.092 (range 0.022-0.155) respectively. There was a positive correlation between the fetal drug concentration (r = 0.59, P = 0.001), amniotic fluid drug concentration (r = 0.47, P = 0.013), amniotic fluid/maternal ratio (r = 0.536, P = 0.003) and fetal/maternal ratio (r = 0.72, P < 0.001) with advancing gestational age. CONCLUSIONS: Although naproxen can cross the placenta readily in the first trimester of human pregnancy, only a small amount was present in fetal tissues. Since there is no information on whether this small amount of naproxen would be teratogenic or not, women of reproductive age who are taking naproxen regularly should be warned of the possible fetal side-effects.     

Determination of lamotrigine in human plasma by high-performance liquid chromatography.

The method involves precipitation of plasma proteins with acetonitrile and analysis of the supernatant by high-performance liquid chromatography using a 5 microm Zorbax C8 column. Quantitation was performed by measurement of the UV absorbance at a wavelength of 306 nm. The method was linear in the range of 1-20 microg/ml, with a mean coefficient of determination (r2=0.998). The limit of detection was 0.6 microg/ml and the lower limit of quantitation was 1 microg/ml using 200 microl of plasma. Within- and between-day accuracy and precision were below 6% at all analysed concentrations except at the limit of quantitation. No interfering peaks were found by commonly monitored antiepileptic drugs. Recovery was found to be > or =99%. Satisfactory performance was obtained in the evaluation of epileptic patient samples, whose results of plasma concentration measurements are briefly discussed. We conclude that this is a reliable method for the routine monitoring of lamotrigine concentration in plasma in the clinical setting.     

Evaluation of imipenem/imipenem+EDTA disk method for detection of metallo-beta-lactamase-producing Klebsiella pneumoniae isolated from blood cultures

The objective of this study was to evaluate the imipenem (IMP) and IMP+EDTA (IMP/IMP+EDTA) disk method for the detection of metallo-beta-lactamases (MBLs) in clinical isolates of Klebsiella pneumoniae with various MIC levels to IMP. Forty-one blood isolates of K. pneumoniae with MIC to IMP ranging from < or =0.5 to > or =16 microg/ml were examined. The MICs were determined by VITEK-2 (bioMerieux Vitek two, France). Disks of 10 microg IMP with and without the addition of 0.5 M EDTA were used for the IMP/IMP+EDTA disk method. The E-test (AB Biodisk, Solna, Sweden) for MBL detection was also used. All isolates were examined for the bla (VIM-1) gene by PCR and for clonality of VIM-1-producing isolates by pulsed-field gel electrophoresis (PFGE). All isolates with MIC values of IMP < or =0.5 microg/ml exhibited no differences in inhibition zone diameters (IZD) produced by IMP and IMP+EDTA disks, whereas the isolates with MICs > or =1 microg/ml showed an increase in IZD, ranging from 8 to 26 mm. All isolates with MIC values of > or =1 microg/ml were found positive for the bla (VIM-1) gene by PCR and for MBL production by the E-test, whereas none of isolates with MICs <0.5 microg/ml was found positive by any of the tests. DNA restriction fragments generated by PFGE of VIM-1-producing isolates were classified in four main types. The IMP/IMP+EDTA disk method is simple to perform, sensitive, and specific for detection of MBL-producing K. pneumoniae clinical isolates. K. pneumoniae isolates with MICs of IMP > or =1 microg/ml and/or IZD produced by IMP disk <19 mm should be tested for MBL production.     

New sensitive assay of vancomycin in human plasma using high-performance liquid chromatography and electrochemical detection

A method using reversed-phase high-performance liquid chromatography with electrochemical detection for the analysis of vancomycin in human plasma was developed. Chromatographic conditions included an octadecyl column, a mobile phase of acetonitrile-sodium phosphate buffer (pH 7) (12:88), a total run time of 12 min, and coulometric electrochemical detection at +700 mV. Linear detector response was found in the range 5-100 microg ml(-1) after a 1:80 dilution or from 0.5 to 50 microg ml(-1) after a 1:20 dilution of the samples. In both cases the correlation coefficient (r) of the calibration curve standard was better than 0.995. Vancomycin determination was based on a denaturation of plasma proteins with methanol, then a dilution with mobile phase was performed. Recovery of vancomycin from plasma was 103.1+/-3.9%, and no interference from commonly used drugs or endogenous compounds was observed. A significant correlation was shown with the EMIT assay (r=0.92, P<0.001) using clinical samples from children. This HPLC technique is simple, sensitive, rapid, precise, selective and requires only 100 microl of plasma for completion.     

Liquid chromatographic-mass spectrometric determination of the novel, recently identified thioTEPA metabolite, thioTEPA-mercapturate, in urine.

An assay for the quantitative determination of the mercapturic acid conjugate of N,N',N"-triethylenethiophosphoramide (thioTEPA-mercapturate) in human urine has been developed. ThioTEPA-mercapturate, a recently identified metabolite of the alkylating anticancer agent thioTEPA, was analyzed using LC-MS and with direct sample injection. Sulphadiazine was used as internal standard. Linearity was accomplished in the therapeutic relevant range of 1-25 microg/ml; recovery was 84% and both accuracy and precision were less than 20% for the lower limit of quantification (1.0 microg/ml) and less than 10% for the other concentration levels. The stability of thioTEPA-mercapturate proved to be satisfactory over a period of 2 months, when kept at -80 degrees C. ThioTEPA-mercapturate urine concentrations of two patients treated with thioTEPA are presented demonstrating the applicability of the assay for clinical samples.     

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 microg/ml using 0.5 ml of serum. The calibration curves from 0.19 to 9.41 microg/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 the cephalosporin antibiotic cephradine in human plasma by high-performance liquid chromatography with ultraviolet detection

A simple and sensitive high-performance liquid chromatographic method, for the determination of cephradine in human plasma samples has been developed and validated. Cephradine and cephaloridine (internal standard) were extracted from human plasma by perchloric acid protein precipitation followed by centrifugation. Aliquots of the extracts were analysed by reversed-phase high-performance liquid chromatography (HPLC) utilising a polymeric reversed-phase PLRP-S column, followed by ultraviolet detection at 260 nm. The method has a working dynamic range from 0.2 to 30.0 microg/ml from 200 microl human plasma. The precision of the method at 0.2 microg/ml was 4.9% (intra-assay) and negligible (inter-assay) as calculated by one-way analysis of variance and the accuracy of the method at 0.2 microg/ml was -4.1% in terms of percentage bias. This method has been successfully applied to clinical studies including an oral bioequivalence study comparing the pharmacokinetics of 500 mg tablets of Kefdrin with 500 mg tablets of Velosef in healthy human volunteers.     

Quantification of busulfan in plasma by liquid chromatography-ion spray mass spectrometry. Application to pharmacokinetic studies in children.

Optimisation of busulfan dosage in patients undergoing bone marrow transplantation is recommended in order to reduce toxic effects associated with high drug exposure. A new method was developed coupling liquid chromatography with mass spectrometry (LC-MS) and was validated for the determination of busulfan concentrations in plasma. Recovery was 86.7%, the limit of detection was 2.5 ng/ml and linearity ranged from 5 to 2500 ng/ml. The correlation between the busulfan concentrations measured by our previously published HPLC-UV method and the new HPLC-MS method was highly significant (P<0.0001). Sample volume was reduced and the method was rapid, sensitive and less expensive than the methods previously used in our laboratory. This method was used to determine the pharmacokinetic parameters of busulfan after the first administration of 1 mg/kg orally, in 13 children receiving the drug as part of the preparative regimen for bone marrow transplantation. Our results were similar to previously reported data. They showed that the apparent oral clearance of busulfan was 0.299+/-0.08 l/h/kg, and that it was significantly higher (P=0.02) in patients below the age of 5 years than in older children.     

Determination of free concentrations of ropivacaine and bupivacaine in plasma from neonates using small-scale equilibrium-dialysis followed by liquid chromatography-mass spectrometry

Attempts to determine a safe plasma concentration of ropivacaine and bupivacaine in neonates have not been consistent. This might be due to an underestimation of free drug in small plasma samples by currently used techniques, e.g., ultrafiltration. We describe a simple microscale equilibrium-dialysis technique for the separation of free and bound ropivacaine and bupivacaine. The free drug in the dialysate was determined using solid-phase extraction and liquid chromatography with mass spectrometry. Pentycaine was used as an internal standard and added to the dialysates prior to extraction. The method is very selective and sensitive, as no compounds other than the analyte and internal standard were observed in the resulting chromatograms at low ng/ml levels. The limit of quantitation was 2.5 ng/ml. The calibration curve was linear in the range of 2 to 1000 ng/ml. The precision of the whole procedure was 8.1% (n=10) and 6.5% (n=7) for ropivacaine and bupivacaine, respectively. The method was tested in the analysis of plasma samples taken from neonates who had received epidural injections.     

Detection of nevirapine in plasma using thin-layer chromatography

BACKGROUND: Nevirapine (NVP) is widely prescribed in resource-poor settings to pregnant women for treatment and prevention of HIV infection. High rates of misreported adherence, however, have compelled clinicians to find alternative methods to ensure systemic drug exposure. This report describes a fast, inexpensive thin-layer chromatography (TLC) method to detect the presence of NVP in human plasma. METHODS: Human plasma was spiked with various concentrations of NVP. NVP was subsequently isolated using solid-phase extraction and visualized with TLC. Clinical samples with NVP concentrations predetermined by high-performance liquid chromatography were used to validate the TLC method. RESULTS: NVP was detected at concentrations as low as 60 ng/mL. The lower limit of detection was set at 100 ng/mL due to the clear spot definition at this concentration. The turnaround time for assay results averages several hours, and costs associated with the assay are considerably below standard drug quantitation techniques. CONCLUSION: TLC provides a rapid, sensitive, and economical tool to qualitatively measure NVP in plasma. This method offers clinicians in resource-poor settings an alternative approach for measuring adherence, particularly in developing-world regions where NVP use is common and there is an immediate need to prevent mother-to-child HIV transmission.     

Determination of acrylamide and glycidamide in rat plasma by reversed-phase high performance liquid chromatography

Acrylamide is a widely used monomer that produces peripheral neuropathy. It is metabolized to the epoxide, glycidamide, which is also considered to be neurotoxic. A new reversed-phase high-performance liquid chromatography (HPLC) method is described that permits simultaneous determination of acrylamide and glycidamide in rat plasma. Samples were deproteinized with acetonitrile and chromatography was performed using isocratic elution and UV absorption detection. The limits of detection for acrylamide and glycidamide were 0.05 and 0.25 microg/ml in plasma, respectively, and recovery of both analytes was greater than 90%. The assay was linear from 0.1 to 100 microg/ml for acrylamide and from 0.5 to 100 microg/ml for glycidamide. Variation over the range of the standard curve was less than 15%. The method was used to determine the concentration-time profiles of acrylamide and glycidamide in the plasma of acrylamide-treated rats.     

Evaluation of a new chromogenic medium, MRSA select, for detection of methicillin-resistant Staphylococcus aureus

We compared MRSA Select to mannitol-salt agar with 8 microg/ml cefoxitin for the detection of methicillin-resistant Staphylococcus aureus (MRSA) from 6,199 clinical samples submitted for MRSA screening. The sensitivities and specificities of MRSA Select and mannitol-salt agar with cefoxitin were 98% and 92% versus 90% and 78%, respectively (P<0.0001). Most (96%) MRSA were detected after overnight incubation using MRSA Select.     

Convenient selective differential broth for isolation of vancomycin-resistant enterococcus from fecal material

Studies have shown that vancomycin broth enrichment is superior to direct plating for the detection of vancomycin-resistant enterococcus (VRE), but vancomycin selective broth is not generally commercially available. We developed an easy-to-prepare VRE selective differential broth and compared it to direct plating on bile esculin azide (BEA) agar for the isolation of VRE from fecal samples. A total of 528 consecutive rectal swabs and stools were inoculated onto BEA agar and into BEA broth with vancomycin at a concentration of 15 microg/ml (BEA VAN15 microg/ml broth). After 1 to 2 days of incubation, broths were subcultured to BEA VAN6 microg/ml agar. Bile esculin-positive colonies from the direct and broth subculture plates were evaluated for the presence of VRE by standard microbiological techniques. Addition of the broth enrichment step led to the detection of significantly more VRE isolates than did direct plating alone (28 versus 18 VRE isolates, respectively). In all, 30 VRE strains were isolated from 29 cultures, all of which were Enterococcus faecium. MICs of vancomycin ranged from 32 microg/ml (n = 2) to > 256 microg/ml (n = 28). Twenty-two VRE isolates were available for further testing: sixteen exhibited a VanA phenotype and six were of the VanB phenotype. van genotypes were in agreement with phenotypes for all VRE isolates except one, which could not be genotyped. The broth method also resulted in significantly fewer bile esculin-positive, non-VRE isolates requiring further workup. We have thus developed an easily prepared vancomycin selective differential broth that is significantly more sensitive and specific in the detection of VRE than is direct fecal plating to BEA agar.