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PubMed2005

Integrity profiling of high throughput screening hits using LC-MS and related techniques.

Integrity profiling of HTS hits is valuable for verification of the hit identity and purity. This provides early discovery researchers with more confident SAR theories. Methodology for integrity profiling of HTS hits must be high throughput, consume little material, and selectively provide structure-based data. Analytical techniques that can be utilized for integrity profiling methods are reviewed for their appropriateness in sample preparation, component separation, detection, purity quantitation, identity confirmation, and follow-up.

Chromatography, Liquid↗

PubMed1977

A nutrient re-evaluation of shell eggs.

Most data on the nutrient composition of shell eggs were obtained before 1950. Since then, management systems for egg production and analytical methods for many components have changed. Freeze-dried whole egg, yolk and white samples were prepared from eggs obtained from a single source of 15 month old White Leghorns. The sample were packaged under nitrogen and held at -20 degrees C. until analyzed. Conversion factors are included for expressing the amount of each component on a per egg as well as a per 100 g. edible liquid basis. This re-evaluation of nutrient data included total solids, lipid, protein, cholesterol, ash, calories, amino acids, fatty acids, vitamins and minerals.

Amino Acids↗

PubMed2006

[2000 Chinese total dietary study--the dietary arsenic intakes].

OBJECTIVE: To assess the safety of dietary arsenic intakes in different areas in China. METHODS: Using the total dietary study method, the analytical samples were obtained in different areas by food consumption survey, food aggregation, food sampling and preparation. The levels of dietary total and inorganic arsenic were determined by the hydride generation atomic absorption spectrometry and atomic fluorospectrophotometry. The dietary total and inorganic arsenic intakes in different areas were obtained by timing the food consumption data and the arsenic content in different dietary samples. The safety of dietary arsenic was evaluated in Chinese four regions and average adults by using the dietary inorganic arsenic PTWI recommended by WHO. RESULTS: The results indicate that the dietary arsenic intake is safety in different regions. Only a few samples in some areas exceed tolerance limits of China national standard. Dietary total and inorganic arsenic (of PTWI) intakes in four different regions (north1, north2, south1, south2 and average adults) were 0.220 mg and 0.094 mg (69.3%), 0.254 mg and 0.098 mg (72.2%), 0.296 mg and 0.048 mg (35.6%), 0.335 mg and 0.077 mg (57.3%), 0.276 mg and 0.079 mg (58.6%) respectively. Mainly sources of dietary arsenic intake were cereals, vegetables and beverage and water. CONCLUSION: It is the first time that the dietary total arsenic and inorganic arsenic intakes of the adult were investigated in Chinese total diet study. The result shows that the total and inorganic arsenic intakes in Chinese people are safe.

Adult↗

PubMed2006

[Chinese total diet study in 2000--the dietary mercuric intakes].

OBJECTIVE: To assess the safety of dietary mercuric intakes in different areas in China. METHODS: Using the Chinese total dietary study method, the analytical samples were obtained in different areas by food consumption survey, food aggregation, food sampling and preparation. The levels of dietary total mercury were determined by cold vapor atomic absorption spectrometry. The dietary total mercuric intakes in different areas were obtained by timing the food consumption data and the mercuric content in different dietary samples. The safety of dietary mercury was evaluated in Chinese four regions and average adults by using the dietary mercuric PTWI recommended by WHO. RESULTS: The results indicate that the dietary mercuric intakes are safe in different regions. Only a few samples in some areas exceed tolerance limits of China national standard. Dietary total mercuric intakes (of PTWI) in four different regions adults and mean were 8.86 microg (19.7%), 4.69 microg (10.4%), 7.01 microg (15.6%), 6.88 microg (15.3%), 6.86 microg (15.2%), respectively. Mainly sources of dietary mercuric intake were cereals, vegetables and sea foods. CONCLUSION: The result shows that the total mercuric intakes in Chinese people are safe.

Adult↗

PubMed1998

[Solid phase microextraction (SPME) of sample preparation during of a complex biological matrix in biotransformation studies].

Within the scope of the investigation of drug metabolism in keratinocytes solid phase microextraction (SPME) was investigated as a suitable method for sample preparation. The application of SPME is based on the fact, that a amount of analyte is absorbed by the polymer fiber at equilibrium, and the fiber is localized on a tip of a GC-syringe. The stable nitroxyl radical TEMPO (2,2,6,6-tetramthylpiperidine-1-oxyl) and its apolar metabolite 2,2,6,6-tetramethylpiperidine were analyzed by SPME and subsequent GC using thymol as internal standard. By means of the headspace-technique and an apolar fiber the recovery rate of TEMPO and the metabolite was nearly 100% and the precision was high. However, the results of the direct SPME were unsatisfactory. In comparison with conventional liquid/liquid extraction and solid phase extraction SPE the SPME proved the best results with regard to recovery rate and precision. Furthermore, the main advantages of SPME are the renunciation of organic solvents, the saving of time, the possibility to reuse the fiber about 100-150 times and the option for a complete automatisation of the extraction procedure.

Biotransformation↗

PubMed1999

Simultaneous determination of amphetamine, methamphetamine, methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA), and methylenedioxyethylamphetamine (MDEA) enantiomers by GC-MS.

A method is described for the simultaneous determination of the ratio of l- and d-enantiomers of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA) in urine. The assay uses liquid-liquid extraction followed by derivatization with trifluoroacetyl-l-prolyl chloride (l-TPC) and analysis by gas chromatography-mass spectrometry. The assay was developed using prepared samples containing varying concentrations of each of the analytes over a range of percentages of each enantiomer. Results showed the method to provide accurate and reliable results in samples containing > or = 10 ng/mL amphetamine and methamphetamine and > or = 25 ng/mL MDA, MDMA, and MDEA. The assay was used to analyze urine samples from subjects of a controlled MDMA study. Results for each of the eight subjects showed a greater percentage of the l-enantiomer of MDMA initially, and the percentage increased with time postdose. Analysis of the metabolite MDA revealed that the proportion of d-enantiomer was initially greater than the l-enantiomer followed by a gradual increase in the proportion of l-enantiomer until it exceeded the amount of the d-enantiomer. In all cases, the l-MDA exceeded the d-MDA within the first 36 h postdose.

3,4-Methylenedioxyamphetamine↗

PubMed2000

Methylmalonic acid measured in plasma and urine by stable-isotope dilution and electrospray tandem mass spectrometry.

BACKGROUND: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization is robust and allows accurate measurement of both low- and high-molecular weight components of complex mixtures. We developed a LC-MS/MS method for the analysis of methylmalonic acid (MMA), a biochemical marker for inherited disorders of propionate metabolism and acquired vitamin B(12) deficiency. METHODS: We added 1 nmol of the internal standard MMA-d(3) to 500 microL of plasma or 100 microL of urine before solid-phase extraction. After elution with 18 mol/L formic acid, the eluate was evaporated, and butyl ester derivatives were prepared with 3 mol/L HCl in n-butanol at 65 degrees C for 15 min. For separation, we used a Supelcosil LC-18, 33 x 4.6 mm column with 60:40 (by volume) acetonitrile:aqueous formic acid (1 g/L) as mobile phase. The transitions m/z 231 to m/z 119 and m/z 234 to m/z 122 were used in the selected reaction monitoring mode for MMA and MMA-d(3,) respectively. The retention time of MMA was 2.2 min in a 3.0-min analysis, without interference of a physiologically more abundant isomer, succinic acid. RESULTS: Daily calibrations between 0.25 and 8.33 nmol in 0.5 mL exhibited consistent linearity and reproducibility. At a plasma concentration of 0.12 micromol/L, the signal-to-noise ratio for MMA was 40:1. The regression equation for our previous gas chromatography-mass spectrometry (GC-MS) method (y) and the LC-MS/MS method (x) was: y = 1.030 x -0.032 (S(y|x) = 1.03 micromol/L; n = 106; r = 0.994). Inter- and intraassay CVs were 3. 8-8.5% and 1.3-3.4%, respectively, at mean concentrations of 0.13, 0.25, 0.60, and 2.02 micromol/L. Mean recoveries of MMA added to plasma were 96.9% (0.25 micromol/L), 96.0% (0.60 micromol/L), and 94.8% (2.02 micromol/L). One MS/MS system used only overnight (7.5 h) replaced two GC-MS systems (30 instrument-hours/day) to run 100-150 samples per day, with reductions of total cost (supplies plus equipment), personnel, and instrument time of 59%, 14%, and 75%, respectively. CONCLUSIONS: This method is well suited for large-scale MMA testing (> or =100 samples per day) where a shorter analytical time is highly desirable. Reagents are less expensive than the anion-exchange/cyclohexanol-HCl method, and sample preparation of batches up to 100 specimens is completed in less than 8 h and is automated.

Chromatography, Liquid↗

PubMed1990

Measurement of polyethylene glycol 400 in urine by direct-injection high-performance liquid chromatography.

We describe a new "high-performance" liquid chromatographic (HPLC) method for estimating polyethylene glycol 400 (PEG 400) in urine. Direct injection of diluted urine samples onto the column eliminates the necessity for lengthy preparative steps, resulting in considerable saving of both time and chemicals. High analytical recoveries (94-102%) and a rapid rate of analysis are achieved. Results vary linearly with concentrations from 0.2 to 1.6 g/L and the six major components can be individually quantified precisely at all concentrations in this range. This method shows better sample recovery, sensitivity, and reproducibility than does a previously described method. The increased sensitivity should allow the use of lower oral doses of polyethylene glycol 400 in future studies of intestinal permeability.

Chromatography, High Pressure Liquid↗

PubMed2004

Trace determination of anthracyclines in urine: a new high-performance liquid chromatography/tandem mass spectrometry method for assessing exposure of hospital personnel.

Health-care workers handling antineoplastic agents may be exposed to extremely low doses of these drugs. Very sensitive and specific analytical methods are therefore needed for biological monitoring. The aim of this study was to develop and validate a method for trace level determination of doxorubicin, epirubicin, daunorubicin and idarubicin in human urine, using epi-daunorubicin as an internal standard. Solid-phase extraction (SPE) was used for sample preparation. Urine samples were loaded onto Bond Elut C18 cartridges. The analytes were eluted in methylene chloride/2-propanol (1:1, v/v) and then evaporated to dryness. The residue was reconstituted with the mobile phase prior to high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) analysis. Quantitation of each analyte was performed using the multiple reaction monitoring (MRM) method. The urine assay was linear over the range 0.1-2.0 microg/L, with a lower limit of quantification (LLOQ) of 0.10 microg/L for doxorubicin and epirubicin, and 0.03 microg/L for daunorubicin and idarubicin. The respective limits of detection (LODs) were 0.04 and 0.01 microg/L. The precision and accuracy of the assay were determined on three different days. The within-series precision was found to be always less than 13.9% for all the analytes. The overall precision expressed as relative standard deviation (RSD) was always less than 10.6%. The recovery of anthracyclines was assessed at two concentrations of the range tested (0.1 and 2.0 microg/L) and it ranged from 87.7% (daunorubicin) to 102.0% (doxorubicin) and from 79.1% (daunorubicin) to 90.7% (idarubicin) for the lower and the higher level, respectively, with a RSD always less than 9.1%. The uncertainty of the present assay was also evaluated and the combined uncertainty was always less than 20% over all the days of the validation study. This is the first method that makes use of LC/MS/MS for the biological monitoring of occupational exposure to anthracyclines.

Anthracyclines↗

PubMed2003

Miniaturized sample preparation combined with liquid phase separations.

Miniaturized sample preparation methods designed as the sample pretreatment for liquid phase separations, such as liquid chromatography, capillary electrophoresis and capillary electrochromatography, have been reviewed especially for the on-line coupling of the sample preparation process and the separation process. The development of the desorption interfaces for the effective combining of the sample preparation and subsequent liquid phase separations is briefly described along with the applications of the combined analytical systems to the analysis of complex sample mixtures such as biological and environmental matrices. Novel use of fine polymeric filaments as the extraction medium for microscale liquid phase separation methods are investigated and a comparison is made with other sample preparation techniques. Polymer coating onto the fibrous material is also introduced to further develop microscale sample preparation methods with improved extraction performance. Several other microscale sample preparation methods having a potential compatibility to the liquid phase separations are also described for future applications of these techniques.

Chromatography, Liquid↗

PubMed1986

A rapid and reliable method for extraction and assay of tricyclic antidepressant drugs in serum or plasma.

Because of their narrow therapeutic ranges and differing patient tolerances, the monitoring of tricyclic antidepressants (TADs) commonly prescribed for control of endogenous depression is highly desirable for the satisfactory treatment of patients. The majority of methods for analysis of TADs require multiple extractions from plasma with organic solvents, accompanied by obligatory centrifugation steps. We describe a simple procedure for the rapid extraction of serum TADs, with high recovery of the analyte drugs. It involves sample preparation using Analytichem 'BOND-ELUT' C-2 reverse phase columns, and analysis by gas chromatography using a nitrogen-phosphorus detector. The method offers a quick, inexpensive and low-maintenance assay which is applicable to all the common tricyclic class drugs.

Antidepressive Agents, Tricyclic↗

PubMed2004

Development and validation of a highly sensitive gas chromatographic-mass spectrometric screening method for the simultaneous determination of nanogram levels of fentanyl, sufentanil and alfentanil in air and surface contamination wipes.

A highly sensitive gas chromatographic-mass spectrometric (GC-MS) analytical method for the determination of the opioid narcotics fentanyl, alfentanil, and sufentanil in industrial hygiene personal air samples and surface contamination wipes was developed and comprehensively validated. Sample preparation involved a single step extraction of the samples with methanol, fortified with a fixed amount of the penta-deuterated analogues of the opioid narcotics as internal standard. The GC-MS analytical procedure using selected ion monitoring (SIM) was shown to be highly selective. Linearity was shown for levels of extracted wipe and air samples corresponding to at least 0.1-2 times their surface contamination limit (SCL) and accordingly to 0.1-2 times their time weighted average occupational exposure limit (OEL-TWA) based on a full shift 9601 air sample. Extraction recoveries were determined for spiked air samples and surface wipes and were found to be quantitative for both sampling media in the entire range studied. The air sampling method's limit of detection (LOD) was determined to be 0.4 ng per sample for fentanyl and sufentanil and 1.6 ng per sample for alfentanil, corresponding to less than 1% of their individual OEL for a full shift air sample (9601). The limit of quantification (LOQ) was found to be 1.4, 1.2, and 5.0 ng per filter for fentanyl, sufentanil, and alfentanil, respectively. The wipe sampling method had LODs of 4 ng per wipe for fentanyl and sufentanil and 16 ng per wipe for alfentanil and LOQs of respectively, 14, 12, and 50 ng per wipe. The analytical intra-assay precision of the air sampling and wipe sampling method, defined as the coefficient of variation on the analytical result of six replicate spiked media was below 10 and 5%, respectively, for all opioids at all spike levels. Accuracy expressed as relative error was determined to be below 10%, except for alfentanil at the lowest spike level (-13.1%). The stability of the opioids during simulated air sampling was investigated. For fentanyl and sufentanil a quantitative recovery was observed at all spike levels, while for alfentanil recoveries ranged from 60.3 to 85.4%. When spiked air samples were stored at ambient temperature and at -15 degrees C quantitative recovery was found for fentanyl and sufentanil after 7 and 14 days. For alfentanil a slight loss seemed to occur upon storage during 7 days, being more explicit after 14 days. Ambient storage of spiked wipes seemed to lead to significant losses of all opioids studied, yielding recoveries of 37.7-88.3%. Upon storage of similar wipes at -15 degrees C a significantly higher recovery was found ranging from 77.3 to 88.3%. The developed analytical and sampling procedures have been recently applied in an explorative field study of which the results of surface contamination wipe sampling are presented in this paper. To our knowledge, this is the first study addressing the development and validation of analytical procedures for the assessment of external occupational exposure to potent opioid narcotics.

Air↗

PubMed1999

The analysis of cyclophosphamide and its metabolites.

Cyclophosphamide (CP) has been in clinical use for the treatment of malignant disease for over 40 years. CP is inactive until it undergoes complex metabolic pathways leading to the ultimate alkylating agent, phosphoramide mustard, but also to inactive and toxic metabolites. Sensitive and specific methods are now available for the measurement of CP and its enantiomers, its metabolites and their stereoisomers, in biological matrices. An overview is given of the methods of analysis of CP and its metabolites described in literature since 1993 as well as the current knowledge about its metabolism. Five classes of methods are described: (1) thin-layer chromatography-photographic densitometry, (2) high performance liquid chromatography, (3) gas chromatography and gas chromatography coupled to mass spectrometry, (4) phosphorus-31 nuclear magnetic resonance and (5) enantiomeric separation. In each case, sample clean up and preparation are described. Precision and limits of quantification of the assays are indicated. A table summarizes all the analytical methods for assaying each metabolite.

Antineoplastic Agents, Alkylating↗

PubMed2006

A validated method for the determination of traces of UV filters in fish using LC-MS/MS.

An analytical method for the determination of UV filter substances in fish tissue has been developed and validated using benzophenone-3, 3-(4-methylbenzylidene)-camphor, 2-ethylhexyl-2-cyano-3,3-diphenyl-2-propenoate and 2-ethylhexyl 3-(methoxyphenyl)-2-propenoate as target analytes. The fish fillets were homogenised and extracted by Soxhlet extraction. The extracts were run through a clean-up process including gel permeation chromatography followed by solid-phase extraction. Quantification of the compounds was performed using liquid chromatography with tandem mass spectrometric detection. Blank fish as well as spiked blank fish were analysed to validate the analytical method. The analytical method developed has the multiple advantages of enabling separation, simultaneous identification and quantification of each of the four selected compounds in a single run. Contamination of blank samples and abnormally high concentrations in spiked samples were avoided by taking extensive precautions during the fish preparation procedure. The method was validated in accordance with internationally accepted criteria, such as specificity, accuracy and repeatability. The combination of LC with tandem mass spectrometry ensures a high level of specificity. The accuracy of the method was reported as the mean recovery rate for the analytes in the sample matrix. Mean recoveries were in the range 86-108%. The precision is expressed as the relative standard deviation, and in all but one of the cases was 20% or below. The accuracy of the method allows residue analyses to be performed on biological matrices at ng/g levels. The determined limit of quantification for each analyte was 8 ng/g fish. For all spiking levels > or =8 ng/g, relative standard deviations were < or = 20%.

Acrylates↗

PubMed2005

Determination of antiretroviral agents in human serum by capillary electrophoresis.

In this work, a simple and rapid electrokinetic chromatography method for the simultaneous separation of different protease inhibitors (indinavir, ritonavir, saquinavir, nelfinavir), nucleoside reverse transcriptase inhibitors (stavudine, zidovudine, didanosine) and non-nucleoside reverse transcriptase inhibitors (nevirapine, efavirenz) was developed. The analyses were performed in a 75 microm i.d. uncoated fused-silica capillary with 48.5 cm length (effective length of 40 cm) using a running buffer consisting of 20 mmol L(-1) sodium dodecyl sulfate, 10 mmol L(-1) sodium tetraborate, 30% acetonitrile and 5% ethanol. Samples were injected hydrodynamically by applying 50 mbar pressure during 6 s. All analytes were separated within 10 min with a voltage of 20 kV. The proposed method was validated for zidovudine, didanosine and efavirenz in human serum. Serum samples were prepared using a solid-phase extraction procedure (Waters Oasis HLB cartridges). For quantitative purposes, stavudine was chosen as the internal standard (IS). Method validation parameters were determined revealing good migration time repeatability (<0.7% RSD) and peak area repeatability (<1.2% RSD). Intra- and inter-day precisions were less than 1.7% and 4.4% RSD, respectively. Matrix matching analytical curves for each drug were linear in the 1.0-20.0 microg mL(-1) interval (r > 0.998). Limits of detection (LOD) were in range of 0.3-0.5 microg mL(-1). The extraction recoveries were higher than 90% with exception of efavirenz, which was 77.4%. Based on the performance characteristics, the proposed method was found suitable for the determination of zidovudine, didanosine and efavirenz in serum samples.

Acetonitriles↗

PubMed1990

Rapid and simultaneous determination of lactulose and mannitol in urine, by HPLC with pulsed amperometric detection, for use in studies of intestinal permeability.

The lactulose/mannitol dual sugar absorption test is a non-invasive test of intestinal permeability. Its widespread use has been limited by the difficulties of analysis for carbohydrates in urine at low concentrations. We describe a "high-pressure" liquid-chromatographic method for determining lactulose and mannitol in urine, in which anion-exchange chromatography and pulsed amperometric detection are used. Sample preparation is simple and fast, and lactulose and mannitol and the internal standards arabinose and cellobiose are well resolved within 15 min. Analytical response of the method is linear with concentrations to 3 g/L, and one can detect as little as 0.3 mg of lactulose per liter of urine. Analytical recovery was between 90% and 107% for all sugars analyzed, and there was good agreement with results by a gas-chromatographic method (r = 0.993 lactulose, 0.984 mannitol). The method may potentially be applied to the study of other carbohydrates present in biological fluids at low concentrations.

Arabinose↗

PubMed2006

A new on-line sample preparation system for the liquid chromatography/time-of-flight mass spectrometry simultaneous analysis of pesticides in river water.

Current on-line solid-phase extraction methods combined with HPLC for shortening the clean-up operation are not suitable for simultaneously detecting compounds that have a wide variety of hydrophobicities. To solve these problems, we designed a new on-line sample preparation system. The system consists of an eluting pump, a mixing TEE connector, a 10-port 2-position valve and a solid-phase extraction precolumn. The eluate from the precolumn is diluted with a weak solvent from the HPLC at the TEE connection to load low hydrophobic compounds onto the analytical column. The proposed on-line sample preparation system was successfully applied to the simultaneous analysis of 21 pesticides in river water using LC/TOF-MS. In this method, the recoveries from river water samples were 67 to 126% (mean 83%), the reproducibility (CV%) was in the range from 1.1 to 11% (mean 5.6%), the calibration curve was linear in the range from 1 ppb to 500 ppb (r > 0.999) and the detection limits (S/N = 3) were in the range from 0.0034 ppb (daimuron) to 3.3 ppb (oxine-copper).

Automation↗

PubMed2000

Evaluation of ursodeoxycholic acid bioavailability from immediate- and sustained-release preparations using gas chromatography-mass spectrometry and high-performance liquid chromatography.

An improved procedure is presented for the determination of ursodeoxycholic acid (CAS 128-13-2, UDCA) in human plasma and bile after oral administration of UDCA-containing dosage forms. The plasma samples after solid-phase extraction with silica-based C18- and strong anion exchange cartridges were assayed by gas chromatography-mass spectrometry (GC-MS) using selected-ion monitoring. The hexafluoroisopropyl trifluoroacetate ester derivative of UDCA was selected for GC analysis since it is easily and rapidly prepared by a one-step reaction. Biliary UDCA levels were determined by a rapid and simple high-performance liquid chromatographic (HPLC) method with on-line sample purification. This analytical protocol was used to investigate the pharmacokinetic of a new sustained-release capsule of UDCA in comparison with a reference immediate-release preparation after single oral administration. Statistical evaluation of the area under the plasma concentration-time curves indicated that two formulations are equivalent with regard to the amount of drug absorbed. However, pharmacokinetic data showed that with the sustained-release preparation a significantly delayed mean peak plasma level was reached compared with the reference preparation. Moreover, the immediate- and extended-release capsules were found to achieve a comparable degree of biliary enrichment with UDCA.

Adult↗