Journal of Chromatography B (v.794, #1)
Editorial Board (iii).
News Section (N1-N3).
Simultaneous determination of deuterated and non-deuterated α-tocopherol in human plasma by high-performance liquid chromatography by Myriam Richelle; Isabelle Tavazzi; Laurent Bernard Fay (1-8).
Labelled tocopherol is used to evaluate its absorption by biodiscriminating the dietary intake from the endogenous tocopherol pool of subject. A normal-phase high-performance liquid chromatographic method is described for the easy separation and quantification of deuterated (d6) and non-deuterated α-tocopherol. The α-tocopherol isotopomers were extracted from plasma triacylglycerol-rich lipoproteins in hexane, separated by two EC Nucleosil columns in series with a mobile phase of hexane–isopropanol (659.34:0.786, w/w) running isocratically. The detection of d6-α-tocopherol was performed by its UV absorbance at 297 nm with a limit of detection of 34 pmol/ml, a limit of quantification of 83 pmol/ml and a range of determination of 34–9905 pmol/ml. Between- and within-assay RSDs were 2.4% (n=10) and 2.7% (n=5), respectively.
Determination of mifepristone levels in wild canid serum using liquid chromatography by Charles Stith; M. Delwar Hussain (9-15).
An HPLC method was developed to determine levels of mifepristone, in coyote (Canis latrans) serum where mifepristone will be used as an oral contragestive agent for nonlethal predator control. Serum samples were extracted using C18 solid-phase extraction cartridges. A synthetic analog of mifepristone, RTI-3021-003, was used as the internal standard. Separation of the compounds was achieved by using a C18 (150×4.6 mm) column. The mobile phase was 55% acetonitrile in water running at 1.0 ml/min with UV detection at 305 nm. The assay was linear in the range of 10 to 1000 ng/ml. Inter-day accuracies for 10, 200 and 1000 ng/ml were 95.9, 99.4 and 104.7%, respectively. Inter-day precisions measured by RSD were 19.8, 9.7 and 4.5%. Intra-day accuracies were 117, 106.9 and 99.4% for 10, 200 and 1000 ng/ml, respectively. Intra-day RSDs were 19.7, 3.7 and 9.3%, respectively. A simple, sensitive and validated HPLC analytical method was developed to quantitate mifepristone in canine serum.
Determination of vigabatrin by capillary electrophoresis with laser-induced fluorescence detection by Sarah Y. Chang; Wen-Chung Lin (17-22).
A new analytical method for vigabatrin based on capillary electrophoretic separation and laser-induced fluorescence detection has been developed. 5-Carboxytetramethylrhodamine succinimidyl ester was used for precolumn derivatization of the non-fluorescent drug. Optimal separation and detection were obtained with an electrophoretic buffer of 50 mM sodium borate (pH 9.5) containing 10 mM sodium dodecyl sulfate and a green He–Ne laser (excitation at 543.5 nm, emission at 589 nm). The concentration limit of detection in aqueous solution was 24 nM. Combined with a simple cleanup procedure, this method can be applied to the determination of vigabatrin in human plasma. A calibration curve ranging from 1.5 to 200 μM was shown to be linear. Both the within-day and day-to-day reproducibilities and accuracies were less then 14.3% and 4.9% respectively. The limit of detection of vigabatrin in plasma was about 0.13 μM.
Determination of risedronate in human urine by column-switching ion-pair high-performance liquid chromatography with ultraviolet detection by P.T Vallano; S.B Shugarts; W.F Kline; E.J Woolf; B.K Matuszewski (23-33).
An HPLC assay for the determination of risedronate in human urine was developed and validated. Risedronate and the internal standard were isolated from 5-ml urine samples in a two-part procedure. First, the analytes were precipitated from urine along with endogenous phosphates as calcium salts by the addition of CaCl2 at alkaline pH. The precipitate was then dissolved in 0.05 M ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid and subjected to ion-pair solid-phase extraction using a Waters HLB cartridge (1 ml, 30 mg) with 1-octyltriethylammonium phosphate as the ion-pair reagent. Following extraction, the analytes were initially separated from the majority of co-extracted endogenous components on a Waters X-Terra RP18 (4.6×50 mm, 3.5 μm) column. The effluent from the X-Terra was “heart-cut” onto a Phenomenex Synergi Polar RP (4.6×150 mm, 4 μm) column for final separation. UV detection (λ=262 nm) was used to quantitate risedronate in the concentration range of 7.5–250 ng/ml. Mean recovery was 83.3% for risedronate and 86.5% for the internal standard. The intra-day precision of the assay, as assessed by replicate (n=5) standard curves, was better than 6% RSD for all points on the standard curve. Within-day accuracy for the standards ranged from 96.3 to 106.1% of nominal. Inter-day precision for quality controls assayed over a 3-week period was better than 5%, while inter-day accuracy was within 90% of nominal. The assay was employed to analyze samples collected during a clinical pharmacokinetics study.
Analysis of eighteen antidepressants, four atypical antipsychotics and active metabolites in serum by liquid chromatography: a simple tool for therapeutic drug monitoring by Christine Frahnert; Marie Luise Rao; Katja Grasmäder (35-47).
Therapeutic drug monitoring necessitates efficient, fast and reliable analytical methods validated by external quality control. We therefore devised an isocratic reversed-phase HPLC method with ultraviolet detection and optimised this to quantify mirtazapine, reboxetine, moclobemide, venlafaxine, O-desmethylvenlafaxine, paroxetine, fluvoxamine, fluoxetine, norfluoxetine, sertraline, citalopram, amitriptyline, nortriptyline, imipramine, desipramine, doxepin, nordoxepin, clomipramine, norclomipramine, trimipramine, mianserine, maprotiline, normaprotiline, amisulpride, clozapine, norclozapine, quetiapine, risperidone and 9-OH-risperidone in human serum. After solid-phase extraction of the drugs and metabolites, the chromatographic separation was achieved on a Nucleosil 100-Protect 1 column with acetonitrile–potassium dihydrogenphosphate buffer as mobile phase. The method was validated for therapeutic and toxic serum ranges. A linear relationship (r>0.998) was obtained between the concentration and the detector signal. Recoveries were between 75 and 99% for the drugs and metabolites. The accuracy of the quality control samples, expressed as percent recovery, ranged from 91 to 118%; intra- and inter-assay-relative standard deviations were 0.9–10.2% and 0.9–9.7%, respectively. Additional external quality control is carried out since 3 years. This method is applicable to rapidly and effectively analyze serum or plasma samples for therapeutic drug monitoring of about 30 antidepressants and atypical antipsychotics.
Keywords: Antidepressants; Antipsychotics, atypical;
High-performance liquid chromatographic determination of quercetin in human plasma and urine utilizing solid-phase extraction and ultraviolet detection by Kazuo Ishii; Takashi Furuta; Yasuji Kasuya (49-56).
An HPLC method for determining quercetin in human plasma and urine is presented for application to the pharmacokinetic study of rutin. Isocratic reversed-phase HPLC was employed for the quantitative analysis by using kaempferol as an internal standard. Solid-phase extraction was performed on an Oasis™ HLB cartridge (>95% recovery). The HPLC assay was carried out using a Luna ODS-2 column (150×2.1 mm I.D., 5 μm particle size). The mobile phase was acetonitrile–10 mM ammonium acetate solution containing 0.3 mM EDTA–glacial acetic acid, 29:70:1 (v/v, pH 3.9) and 26:73:1 (v/v, pH 3.9) for the determination of plasma and urinary quercetin, respectively. The flow-rate was 0.3 ml/min and the detection wavelength was set at 370 nm. Calibration of the overall analytical procedure gave a linear signal (r>0.999) over a concentration range of 4–700 ng/ml of quercetin in plasma and 20–1000 ng/ml of quercetin in urine. The lower limit of quantification was ∼7 ng/ml of quercetin in plasma and ∼35 ng/ml in urine. The detection limit (defined at a signal-to-noise ratio of about 3) was ∼0.35 ng/ml in plasma and urine. A preliminary experiment to investigate the plasma concentration and urinary excretion of quercetin after oral administration of 200 mg of rutin to a healthy volunteer demonstrated that the present method was suitable for determining quercetin in human plasma and urine.
Determination of dextropropoxyphene and nordextropropoxyphene in urine by liquid chromatography–electrospray ionization mass spectrometry by Hans-Åke Lakso; Åke Norström (57-65).
Dextropropoxyphene and nordextropropoxyphene were extracted from urine samples with mixed mode solid-phase extraction cartridges. After elution and evaporation to dryness, the eluate was dissolved in mobile phase and each sample was injected in a LC–ESI-MS system. Quantification was carried out in the selected ion monitoring mode. This article shows the possibility to analyse drugs of abuse substances in urine with a single quadrupole mass spectrometer if only a thorough work-up procedure and a sufficient chromatographic separation is accomplished. In order to enhance the fragmentation of the analytes, in-source fragmentation was carried out. One fragment and the pseudomolecular ion per analyte together with chromatographic retention times were sufficient to verify that the sought compound was found in the samples. In- and between day variation was lower than 10% and the recovery was well above 90%. The analytes were quantified in the range 100–10 000 ng/ml urine.
Keywords: Dextropropoxyphene; Nordextropropoxyphene;
Purification of penicillin G acylase using immobilized metal affinity membranes by Yung-Chuan Liu; Chih-Chiang ChangChien; Shing-Yi Suen (67-76).
The immobilized metal affinity membrane (IMAM) with modified regeneration cellulose was employed for purification of penicillin G acylase (PGA). For studying PGA adsorption capacity on the IMAM, factors such as chelator surface density, chelating metal, loading temperature, pH, NaCl concentration and elution solutions were investigated. The optimal loading conditions were found at 4 °C, 0.5 M NaCl, 32.04 μmol Cu2+ per disk with 10 mM sodium phosphate buffer, pH 8.5, whereas elution conditions were: 1 M NH4Cl with 10 mM sodium phosphate buffer, pH 6.8. By applying these chromatographic conditions to the flow experiments in a cartridge, a 9.11-fold purification in specific activity with 90.25% recovery for PGA purification was obtained. Meanwhile, more than eight-times reusability of the membrane was achieved with the EDTA regeneration solutions.
Keywords: Penicillin G acylase; Enzymes;
Quantification of 6-deoxy-6-demethyl-4-dedimethylaminotetracycline (COL-3) in human plasma using liquid chromatography coupled with electrospray ionization tandem mass spectrometry by Yu-Luan Chen; Glenn.D Hanson; Naidong Weng; Christopher Powala; Brad Zerler (77-88).
An accurate and reliable liquid chromatographic–tandem mass spectrometric (LC–MS–MS) method has been developed and validated for the determination of 6-deoxy-6-demethyl-4-dedimethylaminotetracycline (COL-3) in human plasma. The assay used chrysin as an internal standard (I.S.). The analyte and the I.S. were extracted from acidified plasma by methyl-t-butyl ether. Separation was achieved on a YMCbasic column using acetonitrile–water–formic acid mobile phase. The MS–MS detection was by monitoring fragmentation 372.1→326.2 (m/z) for COL-3 and 255.1→153.1 (m/z) for the I.S. on a Sciex API 365 using a Turbo Ionspray in positive ion mode. The retention times were approximately 1.7 min for COL-3 and 1.8 min for the I.S. The validated dynamic range was 0.03–10.0 μg/ml using 0.25-ml plasma with correlation coefficients of ≥0.9985. The precision and accuracy for the calibration standards (n=3) were RSD≤5.3% and RE≤4.0%. The precision and accuracy for low-, mid- and high-concentration QC samples were RSD≤2.8% and RE≤5.1% for intra-batch (n=6) and RSD≤2.3% and RE≤3.4% for inter-batch (n=18), respectively. The extraction recoveries were 99% for COL-3 and 93% for I.S. The results showed that the quality control plasma samples were stable for at least 1 year if stored at approximately −70 °C. The presented method is simple, fast, specific and rugged. This method has been successfully used for supporting human pharmacokinetic studies.
Validation of a sensitive assay for thiocoraline in mouse plasma using liquid chromatography–tandem mass spectrometry by Jianming Yin; Pablo Aviles; William Lee; Carl Ly; Maria Jose Guillen; Pilar Calvo; Ignacio Manzanares; Glynn Faircloth (89-98).
A sensitive high-performance liquid chromatography–tandem mass spectrometry assay for thiocoraline, an anti-tumor depsipeptide, in mouse plasma is described. Echinomycin, a quinoxaline peptide, was used as an internal standard. Thiocoraline was recovered from the mouse plasma using protein precipitation with acetonitrile and followed by solid-phase extraction of the supernatant. The mobile phase consisted of methanol (0.1% formic acid)–water (0.1% formic acid) (90:10, v/v). The analytical column was a YMC C18. The standard curve was linear from 0.1 to 50 ng/ml (R 2>0.99). The lower limit of quantitation was 0.1 ng/ml. The assay was specific based on the multiple reaction monitoring transitions at m/z 1157→215 and m/z 1101→243 for thiocoraline and the internal standard, echinomycin, respectively. The mean intra- and inter-day assay accuracies remained below 5 and 12%, respectively, for all calibration standards and quality control (QC) samples. The intra- and inter-day assay precisions were less than 11.4 and 9.5% for all QC levels, respectively. The utility of the assay was demonstrated by a pharmacokinetic study of i.v. (bolus) thiocoraline on CD-1 mice. Thiocoraline was stable in mouse plasma in an ice-water bath for 6 h and for three freeze–thaw cycles. The reconstituted thiocoraline after extraction and drying sample process was stable in the autosampler for over 24 h. The assay was able to quantify thiocoraline in plasma up to 48 h following dose. Pharmacokinetic analysis showed that thiocoraline has distinct pharmacokinetic profiling when dosed in different formulation solutions. The assay is currently used to measure thiocoraline plasma concentrations in support of a project to develop a suitable formulation with a desirable pharmacokinetic profile.
Determination of the enantiomers of ketamine and norketamine in human plasma by enantioselective liquid chromatography–mass spectrometry by Maria Esther Rodriguez Rosas; Sharvil Patel; Irving W Wainer (99-108).
A sensitive enantioselective liquid chromatographic assay with mass spectrometric detection has been developed and validated for the simultaneous determination of plasma concentrations of (R)- and (S)-ketamine, and (R)- and (S)-norketamine. The compounds were extracted from human plasma using solid-phase extraction and then directly injected into the LC–MS system for detection and quantification. Enantioselective separations were achieved on a liquid chromatographic chiral stationary phase based upon immobilized α1-acid glycoprotein (the Chiral AGP column). The separations were achieved using a mobile phase composed of 2-propanol–ammonium acetate buffer (10 mM, pH 7.6) (6:94, v/v), a flow-rate of 0.5 ml/min and a temperature of 25 °C. Under these conditions, the analysis time was 20 min. Detection of the ketamine, norketamine and bromoketamine (internal standard) enantiomers was achieved using selected ion monitoring at m/z 238.1, 224.1 and 284.0, respectively. Extracted calibration curves were linear from 1 to 125 ng/ml per enantiomer for each analyte with correlation coefficients better than 0.9993 and intra- and inter-day RSDs of less than 8.0%. The method was applied to samples from a clinical study of ketamine in pain management.
Keywords: Ketamine; Norketamine;
Purification and characterization of a small (7.3 kDa) putative lipid transfer protein from maize seeds by Mariana S. Castro; Isabel R. Gerhardt; Stefania Orrù; Piero Pucci; Carlos Bloch (109-114).
The present study reports, for the first time in literature, the purification and biochemical characterization of a small basic protein from maize seeds similar to plant lipid transfer proteins-2, named mLTP2. The mLTP2 consists of 70 amino acid residues and has an M r of 7303.83, determined by electrospray ionization mass spectrometry. The primary structure of mLTP2 was determined by automated Edman degradation of the intact protein and peptides obtained from digestions with trypsin and by C-terminal sequencing using carboxypeptidase Y. The mLTP2 exhibits high sequence similarity (51–44% identical positions) with other plant LTP2s previously described.
Keywords: Lipid transfer proteins;
Quantification of melatonin in human saliva by liquid chromatography–tandem mass spectrometry using stable isotope dilution by Kåre Eriksson; Anders Östin; Jan-Olof Levin (115-123).
A method for the determination of melatonin in human saliva has been developed using high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS–MS). Saliva was collected in plastic tubes. 7-D-Melatonin was added as internal standard and the samples were cleaned and concentrated by solid-phase extraction. The limit of detection was 1.05 pg ml−1 and the limit of quantification was 3.0 pg ml−1. The accuracy of the method was ±14% at 5.60 pg ml−1 and ±9% at 19.6 pg ml−1. The precision was ±13% at 6.18 pg ml−1 and ±11% at 31.2 pg ml−1, respectively. Our HPLC–MS–MS method shows a high sensitivity and specificity for melatonin and more reliable results compared with a radioimmunoassay. The chromatographic method has been used to determine the circadian rhythm of melatonin among three nurses working the night shift and a patient suffering from an inability to fall asleep at night.
Development of a liquid chromatography–mass spectrometry method for the high-accuracy determination of creatinine in serum by Peter Stokes; Gavin O’Connor (125-136).
An LC–MS method for the high-accuracy determination of creatinine in serum has been developed and used to provide results for an international measurement evaluation programme (IMEP) and the Comité Consultatif pour la Quantité de Matière (CCQM) international inter-laboratory studies. An assessment of different sample preparation methods, including ion-exchange chromatography, solid-phase extraction, plasma ultrafiltration and ethanol protein precipitation, revealed that no bias or reduced precision was associated with the quicker less extensive clean-up methods, when using liquid chromatography–isotope dilution mass spectrometry (LC–IDMS) for quantitation. A number of different calibration regimes were also investigated. External calibration was shown to provide adequate calibration for most routine analysis with a relative associated expanded uncertainty (k=2) of 6% at the 95% confidence level. The use of a non-isotopically labelled internal standard was shown to improve the relative expanded uncertainty (k=2) to 4%. However, the difference in retention time between the internal standard and the creatinine was such that a matrix interferent produced an observed bias of over 16%. The use of an isotopically labelled internal standard was shown to reduce any bias to less than 0.2% with an expanded uncertainty (k=2) of less than 0.3%. The developed method was then used, in a blind trial organised jointly by IMEP and CCQM, to determine the amount of creatinine in human serum. The method performed well against the established reference method of ion-exchange chromatography followed by derivatisation gas chromatography (GC)–IDMS. The observed difference between the values determined by LC–IDMS and the key comparison reference value (average of all the submitted results) was less than 0.3%. The biggest advantage of the described method is in the speed of analysis. With a chromatographic run time of less than 10 min and sample preparation consisting of a simple protein precipitation, without the need for a derivatisation stage, the analysis is vastly simpler then the conventional GC–IDMS reference method.
New high-resolution mass spectrometric approach for the measurement of polychlorinated biphenyls and organochlorine pesticides in human serum by John R Barr; Vincent L Maggio; Dana B Barr; Wayman E Turner; Andreas Sjödin; Courtney D Sandau; James L Pirkle; Larry L Needham; Donald G Patterson (137-148).
To increase our analytical throughput for measuring polychlorinated biphenyls (PCBs) and organochlorine (OC) pesticides without sacrificing data quality, we have developed and validated a combined PCB/OC pesticide gas chromatography–high-resolution mass spectrometry (GC–HRMS) analysis. In a single GC–HRMS analysis, both selected PCBs and OC pesticides are detected and quantified. Previously, this has been difficult, if not impossible, because of the major difference in masses of the most abundant electron-impact ions. However, we have identified slightly less abundant ions to monitor that allow us to successfully combine these analytes into a single analysis without sacrificing any analytical sensitivity or instrument reliability. Consequently, we have been able to double our analytical throughput by modification of mass spectrometric parameters alone. Our new methodology has been validated against our current GC–HRMS method, which entails using two separate injections, one for PCB analysis and one for OC pesticide analysis. The two methods differ by less than 4% overall, with no systematic bias. We used this method to analyze approximately 350 serum samples over a period of several months. We found that our new method was as reliable in automated, overnight runs as our current method.
Keywords: Polychlorinated biphenyls; Organochlorine pesticides;
Enzyme assay for protein kinase using micellar electrokinetic chromatography with laser-induced fluorescence detection by Masatake Sano; Kyoji Ueno; Hiroshi Kamimori (149-156).
Micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection has been developed for a protein kinase assay. This protein kinase assay could readily determine the phosphorylation activity of substrate peptide kemptide using cAMP-dependent protein kinase (PKA) as a model enzyme. Kemptide and phosphorylated kemptide could be reacted with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescence derivatization reagent for LIF detection by directly adding NBD-F into the PKA enzymatic reaction mixture. These derivatives of substrate and product were separated and detected within the analysis time of 5 min by micellar electrokinetic mode using a mixture of sodium dodecylsulfate and methanol as a running buffer. Good linearity of the peak response of the phosphorylated kemptide was obtained over the range of 1–20 mU/tube of PKA in the assay. The relative standard deviation of the peak areas of the phosphorylated kemptide using 2, 5 and 10 mU/tube of PKA were calculated to <10.4%, indicating that the assay was reproducible. Also, IC50 values of six PKA inhibitors, the K i value and the inhibition pattern of one inhibitor, which were calculated to estimate by the variation of the peak area of the phosphorylated kemptide using 5 mU/tube of PKA, were consistent with the published data. The sensitivity of the assay was higher than that of enzyme-linked immunosorbent assay (ELISA) for PKA phosphorylation activity, as IC50 values, K i value, and the inhibition mechanism of inhibitors could be estimated using one-tenth amounts of PKA, compared with that of ELISA. The MEKC–LIF is expected to be very useful for protein kinase assay and its application to the estimation of inhibitors because this method does not entail experimentally troublesome procedures such as the preparation of antibody or fluorescence-labeled substrate.
Keywords: Enzymes; Protein kinase;
Validation of a simple liquid chromatography assay for creatine suitable for pharmacokinetic applications, determination of plasma protein binding and verification of percent labeled claim of various creatine products by Adam M. Persky; Günther Hochhaus; Gayle A. Brazeau (157-165).
Creatine has been quantified in various tissues by a range of methodologies. This paper reports on the development and validation of a simplified HPLC assay to determine plasma creatine, plasma protein binding of creatine, creatine in microdialysate and creatine in over-the-counter products. An isocratic, reversed-phase (C18) HPLC assay, using potassium phosphate monobasic (pH 4) as a mobile phase, was validated in human plasma and microdialysis perfusion fluid (normal saline). The lower limit of quantification for the assay was 1 mg l−1 in saline and 5 mg l−1 in plasma. The RSD was below 6% and accuracy was below 12% in both matrices. Protein binding in human plasma was found to be negligible (<10%). Over-the-counter creatine monohydrate products tested contained 100% creatine monohydrate. This assay was found to be suitable for pharmacokinetic studies and the assessment of plasma creatine and skeletal muscle microdialysate.
Simultaneous determination of JTT-501 and its main metabolite in human plasma by liquid chromatography–ionspray mass spectrometry by M. Breda; G. Basileo; C.A. James (167-174).
An LC–MS–MS analytical method was developed for the determination of a new antidiabetic agent, JTT-501 and its main metabolite (JTP-20604) in human plasma. The compounds were isolated from plasma by protein precipitation before analysis by HPLC with atmospheric pressure positive ionisation MS–MS detection. An isotopically labelled analog of JTT-501 was used as the internal standard. Linearity was demonstrated over the calibration range of about 5–10 000 ng/ml for both compounds. The assay was validated with respect to accuracy, precision and analyte stability. This method was used for the determination of plasma concentrations for the two compounds in a clinical tolerability study. A cross-validation exercise between two different mass spectrometers, used for the determination of clinical samples, is also reported.
Keywords: JTT-501; JTP-20604;
Fast and sensitive determination of urinary 1-hydroxypyrene by packed capillary column switching liquid chromatography coupled to micro-electrospray time-of-flight mass spectrometry by Anders Holm; Paal Molander; Elsa Lundanes; Steinar Øvrebø; Tyge Greibrokk (175-183).
The present work reports capillary liquid chromatographic column switching methodology tailored for fast, sensitive and selective determination of 1-hydroxypyrene (1-OHP) in human urine using micro-electrospray ionization time-of-flight mass spectrometric detection. Samples (100 μl) of deconjugated, water diluted and filtered urine samples were loaded onto a 150 μm I.D.×30 mm 10 μm Kromasil C18 pre-column, providing on-line sample clean-up and analyte enrichment, prior to back flushed elution onto a 150 μm I.D.×100 mm 3.5 μm Kromasil C18 analytical column. Loading flow rates up to 100 μl/min in addition to the use of isocratic elution by a mobile phase composition of acetonitrile/water (70/30, v/v) containing 5 mM ammonium acetate provided elution of 1-OHP within 5.5 min and a total analysis time of less than 15 min with manual operation. Ionization was performed in the negative mode and 1-OHP was observed as [M–H]− at m/z 217.08. The method was validated over the concentration range 0.2–40 ng/ml 1-OHP in pre-treated urine, yielding a coefficient of correlation of 0.997. The within-assay (n=6) and between-assay (n=6) precisions were in the range 6.4–7.3 and 7.0–8.1%, respectively, and the recoveries were in the range 96.2–97.5 within the investigated concentration range. The method mass limit of detection was 2 pg, corresponding to a 1-OHP concentration limit of detection of 20 pg/ml (0.09 nmol/l) diluted urine or 0.3 ng/ml (1.35 nmol/l) urine.
On-line coupling of solid-phase extraction with mass spectrometry for the analysis of biological samples by M.W.J van Hout; C.M Hofland; H.A.G Niederländer; A.P Bruins; R.A de Zeeuw; G.J de Jong (185-192).
Solid-phase extraction (SPE) was directly coupled to mass spectrometry (MS) to assess the feasibility of the system for the rapid determination of prednisolone in serum. A C18 stationary phase allowed washing of the cartridge with 25% methanol. Elution was performed by switching the methanol percentage from 25% in the washing step to 50% during elution. The high flow-rates during the extraction (5.0 ml/min) combined with ion-trap MS detection resulted in a total analysis time of 4 min. Some tailing of the prednisolone peak was observed. However, the tailing was found acceptable, since by this elution procedure most matrix compounds were prevented from eluting from the cartridge. Some matrix interference was still observed with a triple-quadrupole MS, even in the multiple reaction monitoring mode. This resulted in a detection limit (LOD) of about 10 ng/ml. The matrix interference and the LOD were similar for atmospheric pressure chemical ionisation and atmospheric pressure photo ionisation. Applying an ion-trap MS in the MS–MS mode resulted in cleaner chromatograms. Due to extensive fragmentation of prednisolone, the LOD was not lower than about 5 ng/ml prednisolone in serum, and a limit of quantitation of about 10 ng/ml (relative standard deviation <15%) was observed.
Application of high-performance liquid chromatography–mass spectrometry to detection of diuretics in human urine by Y. Qin; X.B. Wang; C. Wang; M. Zhao; M.T. Wu; Y.X. Xu; S.Q. Peng (193-203).
A rapid, sensitive and reliable high-performance liquid chromatographic–mass spectrometric method for the detection of 25 diuretics in human urine has been developed. Atmosphere pressure chemical ionization (APCI) and electrospray ionization (ESI) modes were evaluated. A 2-ml volume of urine was extracted under basic conditions and separated on an Agilent Zorbax SB-C18 column (150×2.1 mm, 5 μm). The mobile phase consisted of formic ammonium–formic acid buffer (pH 3.5) and acetonitrile. The effects of capillary temperature, sheath gas pressure and compositions of mobile phase on the sensitivity were studied. The recoveries of most of the diuretics were 75–95%. In the full scan mode, the limits of detection of the 25 diuretics were 0.25–25 ng/ml for APCI and 0.6–250 ng/ml for ESI. Under the optimal conditions, 14 diuretics from authentic urine samples were detected successfully by LC–APCI-MS. To obtain more fragmentation information on the chemical structure for positive confirmation, tandem mass analysis was also investigated.