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PubMed2004

Exploring the importance of the relative solubility of matrix and analyte in MALDI sample preparation using HPLC.

New insight into the role of solubility in the sample preparation process for MALDI MS is reported. Reversed-phase gradient HPLC conditions were developed that enable the analysis of a broad range of analyte polarities with a single method. This HPLC method was used to establish a relative polarity scale for a series of 15 MALDI matrix materials, a set of example peptides, and a series of model polymer materials with a broad range of polarity. Examples of each polymer type within the range of 6000-10,000 were analyzed with six matrixes that cover a broad range of polarity using MALDI TOFMS. With regard to polymer signal-to-noise ratio, the matrix and polymer combinations that had a close match of HPLC retention time produced the best MALDI spectra. Conversely, the matrix and polymer combinations that have a large difference in HPLC retention time produced poor MALDI spectra. The results suggest that there is a relationship between polarity (solubility) and effective MALDI sample preparation. The relative HPLC retention time of an unknown polymer can serve as a starting point for predicting the matrix (or range of matrixes) that would be most effective.

Chromatography, High Pressure Liquid↗
PubMed1996

A confirmatory HPLC-MS/MS method for ten synthetic corticosteroids in bovine urines.

In the present study, an HPLC-MS/MS method to confirm, in bovine urine, the most common synthetic corticosteroids illegally used as growth promoters in livestock breeding will be presented. An API III-Plus (PE-Sciex) triple quadrupole mass spectrometer, interfaced by means of an atmospheric pressure chemical ionization source to the HPLC system, was used. Urine samples were treated with a sulfatase-glucuronidase mixture to cleave the drug-conjugates and then extracted on C18 disposable columns. LC separations were performed on a reversed-phase C18 column with ammonium acetate 0.1 M/acetonitrile (60/40, v/v) as mobile phase. Detection was performed in multiple reaction monitoring mode, negative ions, selecting fragmentations characteristic of 10 corticosteroids used more frequently. Good results, in terms of sensitivity and specificity have been obtained for nine corticosteroids that can be analyzed in the same HPLC run; the limits of sensitivity achieved were 0.05-1.0 ng/ml in urine. Only a more polar corticosteroid, required a different HPLC separation. Practical applications of this technique to real samples proved that it is an effective method to confirm the illegal use of corticosteroids as growth promoter in animal. In comparison with the chemical GC-MS methods the simpler sample preparation and the faster time of analysis permit a considerable increase of sample testing per day without compromising on analytical sensitivity and specificity.

Adrenal Cortex Hormones↗
PubMed1991

Quantitative proton magnetic resonance spectroscopic determination of nifedipine and its photodecomposition products from pharmaceutical preparations.

A rapid (5 min), accurate, and precise (SD 0.51%) analytical method involving 1H NMR spectroscopy for determination of nifedipine and its photodecomposition products in pharmaceutical preparations is described. Analysis of commercial samples by 1H NMR and USP NF XVII procedures has indicated a maximum difference of 1.0%. The 1H NMR method is recommended for analysis of nifedipine since it provides sufficiently accurate results, together with a unique spectrum as a confirmatory identification of nifedipine, its photodecomposition products, and other impurities.

Magnetic Resonance Spectroscopy↗
PubMed2006

HPLC determination of omeprazole in human plasma using a monolithic column.

A rapid and sensitive HPLC method using a monolithic column has been developed for quantification of omeprazole (CAS 73590-58-6) in plasma. The method was specific and sensitive with a quantification limit of 10 ng/ml. Sample preparation involves simple, one-step extraction procedure and analytical recovery was complete. The separation was carried out in reversed-phase conditions using a Chromolith Performance (RP-18e, 100 x 4.6 mm) column with an isocratic mobile phase consisting of 0.01 mol/l disodium hydrogen phosphate buffer-acetonitrile (73:27 v/v) adjusted to pH 7.1. The wavelength was set at 302 nm. The calibration curve was linear over the concentration range 20-1500 ng x ml(-1). The coefficients of variation for inter-day and intra-day assay were found to be less than 7%.

Adult↗
PubMed2001

[Identification system for Sildenafil in health foods].

A substantially available identification system for Sildenafil in health foods was established using 3 different analytical methods; i.e. TLC, preparative TLC/MS and HPLC/photo-diode array. Sildenafil in health foods was extracted with ethyl acetate under alkaline conditions as sample solutions for TLC and preparative TLC, and also extracted with 50% methanol and then diluted with solution of HPLC mobile phase for HPLC. The sample solution for TLC was applied to Silica gel 60 F254 plates with chloroform/methanol/28% ammonia (90:1:5, under layer) as mobile phase. Spots were located under UV radiation at 254 nm and 366 nm, and spraying dragendorff reagent. The conditions for preparative TLC were the same as these of TLC method, and samples abtained from preparative TLC were determined by MS with APCI interface, under both positive and negative modes. The HPLC analysis was carried out on a column of Cosmosil 5C18-AR (4.6 mm x 150 mm, 5 microns) with 0.05 mol/l phosphate buffer pH 3.0/acetonitrile(73:27) as mobile phase and the eluate was monitored by a photo-diode array detector. The quantitative analysis was available, when the peak of this sample on HPLC was detected at 290 nm. When this system was applied to commercial health foods, Sildenafil was identified and their contents were 25 mg-45 mg/tablet or bottle. These contents nearly correspond to that in Viagra, 25 mg, 50 mg/tablet. Therefore, there is a fear of side effects for Sildenafil, when it is taken as health foods.

Chromatography, High Pressure Liquid↗
PubMedDate not supplied

Direct methods for detecting picorna-like virus from dead and alive triatomine insects.

In this work we report four different destructive and non-destructive methods for detecting picorna-like virus particles in triatomines. The methods are based on direct observation under transmission electron microscope and they consist of four ways to prepare samples of presumable infected material. The samples are prepared processing dead or alive insect parts, or even dry or fresh insect feces. The methods can be used as analytical or preparative techniques, for quantifying virus infection and checking virus integrity as well. In this work the four methods are applied in order to detect Triatoma virus (TrV) particles in T. infestans colonies.

Animals↗
PubMed2006

Analysis of acrylamide in coffee and cocoa by isotope dilution liquid chromatography-tandem mass spectrometry.

An accurate and precise method for the quantification of acrylamide using stable isotope dilution liquid chromatography-tandem mass spectrometry was developed and used to measure acrylamide in coffee and cocoa samples. The sample preparation involved extraction of the analyte and its internal standard, 13C3-acrylamide, into water and subsequent defatting of the aqueous extract with dichloromethane. An aliquot of the resulting aqueous extract was then azeotropically dried under reduced pressure and subsequently purified using an aminopropyl-bonded silica cartridge. The purified extracts were then chromatographed on a 5-microm 2.1 x 150 mm Hypercarb column, the effluent of which was monitored for the analyte and its internal standard using positive-ion APCI-selected reaction monitoring. The intra-laboratory reproducibility of the method, expressed as a relative coefficient of variation (%, n=5), was determined at four levels of concentration (12.3, 42.3, 139.3 and 464.8 microg kg(-1)) and was found to vary between 0.6-2.5%. The accuracy of the method was assessed using a reference sample of coffee. The average result obtained using our method differed from the assigned value of the reference material by less than 1%. An analysis of a cocoa sample revealed that the method is capable of precisely estimating acrylamide in challenging matrices down to a level of at least 12.3 microg kg(-1).

Acrylamide↗
PubMed2003

Rapid analysis of fluoxetine and its metabolite in plasma by LC-MS with column-switching approach.

A rapid and sensitive method was developed for the simultaneous determination of fluoxetine and its primary metabolite, norfluoxetine, in plasma. It was based on a column-switching approach with a precolumn packed with large size particles coupled with a liquid chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS). After a simple centrifugation, plasma samples were directly injected onto the precolumn. The endogenous material was excluded thanks to a high flow rate while analytes were retained by hydrophobic interactions. Afterwards, the target compounds were eluted in back flush mode to an octadecyl analytical column and detected by ESI-MS. The overall analysis time per sample, from plasma sample preparation to data acquisition, was achieved in less than 4 min. Method performances were evaluated. The method showed good linearity in the range of 25-1000 ng mL(-1) with a determination coefficient higher than 0.99. Limits of quantification were estimated at 25 ng mL(-1) for fluoxetine and norfluoxetine. Moreover, method precision was better than 6% in the studied concentration range. These results demonstrated that the method could be used to quantify target compounds. Finally, the developed assay proved to be suitable for the simultaneous analysis of fluoxetine and its metabolite in real plasma samples.

Administration, Oral↗
PubMed1999

Automated high-performance liquid chromatographic method with precolumn reduction for the determination of ubiquinol and ubiquinone in human plasma.

We developed a gradient HPLC method with automated precolumn reduction for direct electrochemical detection of ubiquinol-10 (CoQ10H2) and total coenzyme Q10 (TQ10) in human plasma. The concentration of ubiquinone-10 (CoQ10) was calculated by subtraction of CoQ10H2 from TQ10. Preparation of reducing agent and precolumn reduction was performed by a programmable auto-injector. The two mobile phases used were: A, 100% of methanol containing 50 mM sodium perchlorate and 10 mM perchloric acid; and B, a mixture of ethanol and tert.-butanol (80:20, v/v). Sample preparation was simply a deproteinisation process with 10-fold ethanol. A good linear relationship was obtained for CoQ10H2 concentration from 0.1 to 3 micromol/l. The detection limit was 2.5 nmol/l with an injection volume of 20 microl. The analytical recovery and reproduciblity were generally >90%. To validate the method, 18 freshly collected plasma samples of normal healthy subjects were analysed. The mean ratio of CoQ10H2/CoQ10 was 93:7. The proposed method is sensitive, reliable and can be used for clinical investigation.

Automation↗
PubMed2001

Quantitative determination of nitrendipine and its metabolite dehydronitrendipine in human plasma using liquid chromatography-tandem mass spectrometry.

A sensitive and high-throughput LC-MS-MS method was developed for simultaneous determination of nitrendipine (NIT) and its major metabolite, dehydronitrendipine (DNIT) in human plasma using nifedipine as the internal standard. Plasma samples were prepared based on a simple liquid-liquid extraction. The extracted samples were analyzed on a Zorbax SB C(18) column interfaced with a triple quadrupole tandem mass spectrometer. Positive atmospheric pressure chemical ionization was employed as the ionization source. The analytes were detected by use of selected reaction monitoring mode. Standard curves were linear (r > or = 0.995) over the concentration range of 0.4-40 ng/mL for NIT and 0.2-20 ng/mL for DNIT. The intra- and inter-run precision was measured to be below 8.5% for NIT and DNIT. The inter-run accuracy was less than 4% for the analytes. The overall extraction recoveries of NIT and DNIT were determined to be about 75% and 78% on average, respectively. The chromatographic run time was approximately 3 min. More than 120 samples could be assayed daily with this method, including sample preparation, data acquisition and processing. The method developed was successfully used to investigate plasma concentrations of NIT and DNIT in a pharmacokinetic study of volunteers who received NIT orally.

Antihypertensive Agents↗
PubMed2003

Biological and environmental monitoring of hospital personnel exposed to antineoplastic agents: a review of analytical methods.

In order to assess occupational exposure of hospital personnel involved in the preparation and administration of antineoplastic drugs, biological and environmental monitoring are essential to identify the main exposure routes and to quantify potential health risks. If workplace contamination cannot be completely avoided, it is of utmost importance to reduce exposure to the lowest possible levels. To this aim, not only do education and training of the exposed subjects play an important role, but accurate standardized sampling techniques and analytical methods are also required. A critical overview of the most significant methods available in the literature is presented and their value is discussed, especially with respect to their sensitivity and specificity. In addition, attention is given to validation procedures and, consequently, to their reliability. The results from the most important surveys carried out at hospital departments are also discussed, with a view to improving both monitoring strategies and moreover working conditions.

Antineoplastic Agents↗
PubMed1999

Tin speciation in the femtogram range in open ocean seawater by gas chromatography/inductively coupled plasma mass spectrometry using a shield torch at normal plasma conditions.

A sensitive method for the determination of ultratrace organotin species in seawater is described. The merits and demerits of derivatization methods using Grignard reagent or sodium tetraethylborate (NaBEt4) were evaluated in terms of derivatization efficiency, applicability to the programmed temperature vaporization (PTV) method, and procedural blanks. The sensitivity of the gas chromatography/inductively coupled plasma mass spectrometry (GC/ICPMS) was improved by more than 100-fold by operating the shield torch at normal plasma conditions, compared with that obtained without using it. The absolute detection limit as tin reached subfemtogram (fg) levels. Furthermore, the detection limit in terms of relative concentration was improved 100-fold by using the PTV method, which enabled the injection of a large sample volume of as much as 100 microL without loss of analyte. When the organotin species in seawater were extracted into hexane with a preconcentration factor of 1000 after ethylation with NaBEt4 and a 100 microL aliquot of the extract was injected into the GC, the instrumental detection limit in relative concentration reached 0.01 pg/L in original seawater. Sources of contamination of organotin species during the sample preparation were examined, and a purification method of NaBEt4 was developed. Finally, the method was successfully applied to open ocean seawater samples containing organotin species at the level of 1-100 pg/L.

Borates↗
PubMed2000

The determination of RWJ-38705 (tramadol N-oxide) and its metabolites in preclinical pharmacokinetic studies using LC-MS/MS.

A rapid and reliable analytical method is described for the simultaneous determination of RWJ-38705 (tramadol N-oxide) and several of its major metabolites in the plasma of Sprague-Dawley rats and Beagle dogs. Sample preparation using solid phase extraction was followed by reversed phase liquid chromatography (LC) coupled with tandem mass spectrometric (MS/MS) detection in the positive ionization mode. The assay was linear for all analytes over concentrations ranging from approximately 6 to 2000 ng/ml. The inter-assay reproducibility was generally less than 15% while accuracy values were within 13% of theoretical. The overall recovery of the analytes ranged from approximately 40 to 64% in rat plasma and 53-75% in dog plasma. This assay has proven to be sensitive, specific and reproducible, and it has been readily implemented in preclinical PK studies. Representative plasma concentration versus time profiles resulting from administration of TNO to rats and dogs are presented in this communication.

Administration, Oral↗
PubMed1994

A rapid method for the separation and analysis of leaked and liposomal entrapped phosphoramide mustard in plasma.

Pharmacokinetic studies of liposomal drugs should include simultaneous determination of leaked and entrapped drug in biological specimens. Due to the limited stability of many liposomal preparations in biological samples, a rapid analytical procedure is often necessary. Phosphoramide mustard (PM), a key cytotoxic metabolite of a widely used alkylating drug cyclophosphamide, has recently been entrapped into a liposomal formulation and the preparation has been found to be rather unstable in plasma. We have, therefore, developed a rapid method for the separation of liposome-associated PM from the unassociated drug and a method for their quantitation in plasma. This method involves the use of size exclusion mini-gel column and requires minutes to process. Due to the use of internal standards, this method tolerates low recovery and requires the collection of a single fraction of each of liposome-associated PM and the unassociated drug. The recovery of liposomal PM from the first fraction of the gel column was found to be 82.4 +/- 7.9% (SD, n = 8), whereas that of liposome-unassociated PM from the major fraction was 16.8 +/- 2.8% (SD, n = 8). However, the low recovery problem of liposome-unassociated PM was circumvented by adding the internal standard [alpha, beta-2H8] PM prior to separation, thus compensating for the loss of liposome-unassociated PM due to incomplete collection. Two types of standard curve were constructed for quantitation of liposome-associated PM and unassociated PM and the linearity for both was excellent. Assay validation indicated that within-run RSD values at 213 ng, 426 ng and 1065 ng for liposomal PM were 4.2, 4.3 and 3.0%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Analysis of methionine enkephalin in human pituitary by multi-dimensional reversed-phase high-performance liquid chromatography, radioreceptor assay, radioimmunoassay, fast atom bombardment mass spectrometry, and mass spectrometry-mass spectrometry.

Methionine enkephalin (ME = YGGFM) was measured in five individual human post-mortem pituitaries using four different analytical methods, with the objective of comparing the molecular specificities of the methods. Radioreceptor assay (RRA) used a receptor-rich preparation from brain and [3H]etorphine as radioligand to determine ME-like receptoractivity (ME-LR). Radioimmunoassay (RIA) measured ME-like immunoreactivity (ME-LI). Pituitary samples analyzed by RRA and RIA were purified first with a high-performance liquid chromatography (HPLC) gradient on a polymer analytical column. Fast atom bombardment mass spectrometry (FAB-MS) in two different detection modes quantified ME using the protonated molecular ion MH+ of ME at 574 a.m.u. and B/E linked-field selected reaction monitoring (SRM) to monitor the specific unimolecular metastable transition that produced the unique amino acid sequence-determining tetrapeptide fragment ion YGGFA+ from the MH+ precursor ion. Both FAB-MS methods used the deuterated internal standard YGG[2H5-F]M. Samples analyzed with FAB-MS were purified first with multi-dimensional reversed-phase HPLC. The first dimension was an ODS gradient, and the second dimension was a polymer isocratic elution. The following ME amounts were measured (mean +/- standard error of the mean): ME-LR, 7.0 +/- 1.9 micrograms g-1 tissue; ME-LI, 1.8 +/- 0.7 micrograms g-1 tissue; MH+, 2.7 +/- 0.6 micrograms g-1 tissue; SRM, 3.0 +/- 0.8 micrograms g-1 tissue. The FAB SRM method provided the highest level of molecular specificity amount these four analytical methods used to measure picomole amounts of endogenous ME in a human pituitary.

Adult↗
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↗
PubMed2000

Laboratory database to manage electrophoresis and chromatography separations and the associated samples.

A database was developed to store, organize, and retrieve the data associated with electrophoresis and chromatography separations. It allows laboratories to store extensive data on separation techniques (analytical and preparative). The data for gel electrophoresis includes gel composition, staining methods, electric fields, analysis, and samples loaded. The database stores data on chromatography conditions, the samples used, and the fractions collected. The data structure of this database was designed to maintain the link between samples (including fractions) from chromatography separations and their analysis by gel electrophoresis. The database will allow laboratories to organize and maintain a large amount of separation and sample data in a uniform data environment. It will facilitate the retrieval of the separation history of important samples and the separation conditions used.

Chromatography↗
PubMed2000

A RIA combined with SPE for the determination of a dual D2-receptor and beta2-adrenoceptor agonist, AR-C68397XX, in human plasma.

A radioimmunoassay has been developed for the determination of AR-C68397XX, a dual D2-receptor and beta2-adrenoceptor agonist, in human plasma. The method incorporates solid phase sample extraction and is suitable for the determination of the analyte at pg ml(-1) concentrations. The antiserum was raised in Suffolk cross sheep following primary and booster immunisations with an immunogen prepared by conjugating a carboxyphenylmethyl derivative of AR-C68397XX, to bovine serum albumin. The radioligand was prepared by the 125I-labelled iodination of a derivative of AR-C68397XX. The solid phase extraction procedure, using octadecyl sorbent, was introduced to remove matrix interferences in the plasma and to enhance method sensitivity. The calibration range is 20-500 pg ml(-1), using 0.5 ml of undiluted human plasma sample.

Adrenergic beta-2 Receptor Agonists↗