A new strategy implementing mass spectrometry in the diagnosis of congenital disorders of N-glycosylation (CDG).

Objectives: Congenital disorders of N-glycosylation (CDG) are a large group of rare metabolic disorders caused by defects in the most common post-translational modification of proteins. CDGs are often difficult to diagnose as they are manifested with non-specific symptoms and signs. Analysis of serum transferrin (TRF) isoforms, as the classical procedure used to identify a CDG patient, enables to predict pathological steps in the N-linked glycosylation process. Methods: We devised a new strategy based on liquid chromatography-mass spectrometry (LC-MS) for the analysis of TRF isoforms by combining a simple and fast sample preparation with a specific chromatographic cleanup/separation step followed by mass-spectrometric measurement. Single TRF isoform masses were obtained through reconstruction of multiply charged electrospray data collected by quadrupole-MS technology. Hereby, we report the first analyzed serum samples obtained from 20 CDG patients and 100 controls. Results: The ratio of desialylated isoforms to total TRF was calculated for patients and controls. CDG-Type I patients showed higher amounts of bi-sialo isoform (range: 6.7-29.6%) compared to controls (<5.5%, mean percentage 3.9%). CDG-Type II pattern showed an increased peak of tri-sialo isoforms. The mean percentage of tri-sialo-TRF was 9.3% (range: 2.9-12.9%) in controls, which was lower than that obtained from two patients with COG5-CDG and MAN1B1-CDG (18.5 and 24.5%). Intraday and between-day imprecisions were less than 9 and 16%, respectively, for bi-sialo- and less than 3 and 6% for tri-sialo-TRF. Conclusions: This LC-MS-based approach provides a simple, sensitive and fast analytical tool for characterizing CDG disorders in a routine clinical biochemistry while improving diagnostic accuracy and speeding clinical decision-making.

Development and validation of LC-MS/MS method for imatinib and norimatinib monitoring by finger-prick DBS in gastrointestinal stromal tumor patients.

The introduction of imatinib, an oral tyrosine kinase inhibitor, as first-line standard therapy in patients with unresectable, metastatic, or recurrent gastro-intestinal stromal tumor (GIST), strongly improved their treatment outcomes. However, therapeutic drug monitoring (TDM) is recommended for this drug due to the large inter-individual variability in plasma concentration when standard dose is administered. A Cmin higher than 760 ng/mL was associated with a longer progression free survival. Thus, a LC-MS/MS method has been developed and fully validated to quantify imatinib and its active metabolite, norimatinib, in finger-prick dried blood spot (DBS). The influence of hematocrit, sample homogeneity, and spot size and the correlation between finger-prick and venous DBS measurements were also assessed. The method showed a good linearity (R2 > 0,996) between 50-7500 ng/mL for imatinib and 10-1500 ng/mL for norimatinib. Analytes were extracted from DBS samples by simply adding to 3 mm-discs 150 µL of acidified methanol containing IMA-D8. The collected extract was then injected on a LC Nexera system in-house configured for the on-line cleanup, coupled with an API-4000 QT. The chromatographic separation was conducted on a Synergi Fusion-RP column (4 µm, 2x50 mm) while the trapping column was a POROS R1/20 (20 µm, 2x30 mm). The total run time was 8.5 min. DBSs stored at room temperature in plastic envelopes containing a silica-gel drying bag were stable up to 16 months. The proposed method was applied to 67 clinical samples, showing a good correlation between patients' finger-prick DBS and plasma concentrations, measured by the reference LC-MS/MS method, internally validated. Imatinib and norimatinib concentrations found in finger-prick DBS were adjusted by hematocrit or by an experimental correction factor to estimate the corresponding plasma measurements. At the best of our knowledge, the proposed LC-MS/MS method is the first analytical assay to measure imatinib and norimatinib in DBS samples.

Development of a fast LC-MS/MS protocol for combined measurement of six LSDs on dried blood spot in a newborn screening program.

New treatment options and improved strategies for Lysosomal Storage Disorders (LSDs) diagnosis on dried blood spot (DBS) have led to the development of several pilot newborn screening programs. Building on a previously published protocol, we devised a new 6-plex assay based on a single DBS punch incubated into a buffer containing a combination of substrates (GAA, GLA, ASM, GALC, ABG and IDUA). This new protocol incorporates a new trapping and clean-up procedure using perfusion chromatography connected on-line with an analytical column for analyte separation, after enzymatic reaction. Results are available after 4.5 min. Several incubation times were tested in order to reduce sample preparation times and to improve accuracy and reproducibility, also regarding the quenching of the reaction within the time window of linear product accumulation. The collected data demonstrate that an incubation time of 4 h is enough to achieve good reaction efficiency without any impact on sensitivity. The method proved versatile and robust for various instrument configurations. The fast sample preparation and running times allow a high sample throughput; an advantage in newborn screening procedures. This method can also be used for diagnostic purposes, allowing a rapid diagnosis in a few hours.

Practical fluorimetric assay for the detection of anticancer drug SN-38 in human plasma.

The implementation of therapeutic drug monitoring in the routine clinical practice in oncology is mainly limited by the lack of therapeutic indexes for the majority of the anticancer drugs, and by the absence of suitable analytical tools, which can accurately quantify in real time the concentration of the administered drugs and their relevant metabolites in biological fluids. In this work, a simple and efficient fluorimetric determination of SN-38, the active metabolite of the anticancer drug irinotecan, was developed and applied to human plasma samples. The intrinsic fluorescence of SN-38 allowed its quantification in the range 10-500 ng mL-1 with a LOQ of 5.0 ng mL-1 and a LOD of 1.5 ng mL-1. Low interferences due to main metabolites of irinotecan and comedications, commonly associated with administration of irinotecan, were observed. A validation study, according to FDA and EMA guidelines for bioanalytical method validation, was carried out and, finally, blind samples were analyzed in parallel with a HPLC-MS method obtaining an excellent agreement between the two techniques.

Enzyme-Based Electrochemical Biosensor for Therapeutic Drug Monitoring of Anticancer Drug Irinotecan.

Therapeutic drug monitoring (TDM) is the clinical practice of measuring pharmaceutical drug concentrations in patients' biofluids at designated intervals, thus allowing a close and timely control of their dosage. To date, TDM in oncology can only be performed by trained personnel in centralized laboratories and core facilities employing conventional analytical techniques (e.g., MS). CPT-11 is an antineoplastic drug that inhibits topoisomerase type I, causing cell death, and is widely used in the treatment of colorectal cancer. CPT-11 was also found to directly inhibit acetylcholine esterase (AChE), an enzyme involved in neuromuscular junction. In this work, we describe an enzymatic biosensor, based on AChE and choline oxidase (ChOx), which can quantify CPT-11. ACh (acetylcholine) substrate is converted to choline, which is subsequently metabolized by ChOx to give betaine aldehyde and hydrogen peroxide. The latter one is then oxidized at a suitably polarized platinum electrode, providing a current transient proportional to the amount of ACh. Such an enzymatic process is hampered by CPT-11. The biosensor showed a ∼60% maximal inhibition toward AChE activity in the clinically relevant concentration range 10-10 000 ng/mL of CPT-11 in both simple (phosphate buffer) and complex (fetal bovine serum) matrixes, while its metabolites showed negligible effects. These findings could open new routes toward a real-time TDM in oncology, thus improving the therapeutic treatments and lowering the related costs.

Analysis of organo-chlorine pesticides residue in raw coffee with a modified "quick easy cheap effective rugged and safe" extraction/clean up procedure for reducing the impact of caffeine on the gas chromatography-mass spectrometry measurement.

The control of pesticide residues on raw coffee is a task of great importance due to high consumption of this beverage in Italy and in many other countries. High caffeine content can hamper extraction and measurement of any pesticide residue. A tandem extraction protocol has been devised by exploiting the quick easy cheap effective rugged and safe (QuEChERS) scheme for extraction, coupled to a dispersive liquid-liquid micro-extraction (DLLME) in order to drastically reduce caffeine content in the final extract. Gas chromatography-mass spectrometry (GC-MS) has been used for quantification of organo-chlorine pesticides in single ion monitoring (SIM) mode. Method has been validated and performances meet the criteria prescribed by European Union regulations.

Screening of lysosomal storage disorders: application of the online trapping-and-cleanup liquid chromatography/mass spectrometry method for mucopolysaccharidosis I.

In recent years, new treatments have become available to treat some lysosomal storage disorders (LSDs) and many studies suggest that there is a benefit with starting therapy early. Newborn screening should detect diseases early enough for prompt treatment. Some countries include additional conditions, such as some LSDs, into their newborn screening panels. Mucopolysaccharidosis Type I (MPS I) is an autosomal recessive disorder caused by the deficiency of α-L-iduronidase (IDUA) activity. Currently, enzyme replacement therapy (ERT) or bone marrow transplantation is available and this has raised a growing interest for the development of a newborn screening test. In 2009, we reported a new fast and simplified tandem mass spectrometry-based method for quantifying five enzyme activities on dried blood spots. Here, we describe the inclusion of IDUA activity determination for the simultaneous detection of six lysosomal storage diseases. We have defined reference normal ranges by testing 680 healthy newborns and 240 adults. The assay was checked through three confirmed MPS I patients whose IDUA activity was below the normal range. Reproducibility of the assays has been established by assessing the intra-day and inter-day assay imprecisions. This quick assay has been devised to be implemented in newborn screening by liquid chromatography tandem mass spectrometry.

A reliable and rapid tool for plasma quantification of 18 psychotropic drugs by ESI tandem mass spectrometry.

A simple liquid chromatographic tandem mass spectrometry (LC-MS/MS) method has been developed for simultaneous analysis of 17 basic and one acid psychotropic drugs in human plasma. The method relies on a protein precipitation step for sample preparation and offers high sensitivity, wide linearity without interferences from endogenous matrix components. Chromatography was run on a reversed-phase column with an acetonitrile-H2O mixture. The quantification of target compounds was performed in multiple reaction monitoring (MRM) and by switching the ionization polarity within the analytical run. A further sensitivity increase was obtained by implementing the functionality "scheduled multiple reaction monitoring" (sMRM) offered by the recent version of the software package managing the instrument. The overall injection interval was less than 5.5 min. Regression coefficients of the calibration curves and limits of quantification (LOQ) showed a good coverage of over-therapeutic, therapeutic and sub-therapeutic ranges. Recovery rates, measured as percentage of recovery of spiked plasma samples, were ≥ 94%. Precision and accuracy data have been satisfactory for a therapeutic drug monitoring (TDM) service as for managing plasma samples from patients receiving psycho-pharmacological treatment.

Development of a highly sensitive method for the quantification of estrone and estradiol in serum by liquid chromatography tandem mass spectrometry without derivatization.

Measurement of estrone (E1) and estradiol (E2) values <1 pg/mL (3.7 pmol/L) is necessary for postmenopausal, pediatric and male serum samples. Until now this was rarely reached and only through derivatization which can present problems for estradiol. A very sensitive LC-MS/MS method was developed avoiding derivatization, convenient for large-scale studies. The desired sensitivity and specificity were achieved using ESI negative mode, LLE and a 2D chromatography consisting of a trapping column and a second dimension reverse-phase C8 analytical column. A mixture of an aqueous solution of ammonium fluoride at 0.2mM and methanol was used on the analytical column to further increase the sensitivity. Serum LOQ was <0.5 pg/mL (1.9 pmol/L) for E2 and E1 and recoveries ranged from 95 to 105%. No carry-over was detectable. Inter assay CV's were 4.0% at 21 pg/mL (77 pmol/L) for E2, 7.6% at 25 pg/mL (93 pmol/L) for E1. Comparison with commercial direct estrogen assays (Roche Diagnostics E170 for E2, Bioline RIA for E1) exposed analytical unsuitability (due to a combined lack of sensitivity and specificity) for the assay of male, postmenopausal or pediatric samples.

Development of a fast and simple LC-MS/MS method for measuring the F2-isoprostanes in newborns.

BACKGROUND: Quantification of F(2)-IsoPs isomer has been regarded as the "gold standard" to assess oxidative stress status in various adult and neonatal human diseases. These methods require high amounts of plasma. OBJECTIVE: To develop a fast and simple LC-MS/MS method for measuring F(2)-isoprostanes in newborns. METHODS: A sample of heel blood (0.4 mL) was collected in a tube containing EDTA was collected from 20 term healthy newborns. For measurements, the tandem mass spectrometer has been run in multiple reaction monitoring (MRM) with the electrospray source operating in negative ion mode, and by exploiting the transitions m/z 353.3 > 193.2 for F2-IsoPs and 357.3 > 197.2 for the internal standard d4-8-iso PGF2α. RESULTS: The concentration of F(2)-IsoPs (in pg/mL) in the collected cord bloods was 60.50 ± 25.04 (mean ± S.D.). No statistical difference was found between male (57.09 ± 19.69) and female (64.67 ± 31.13) concentrations. The overall efficiency of the extraction has been over 80%, while the recovery on spiked samples has been around 94% for spikes of 100 pg/mL with a C.V. of 7.7%. CONCLUSIONS: We developed a suitable method for large-scale studies with a reduced sample requirement as it is mandatory in neonatal age. Small samples and quick answers are very useful in Neonatology allowing early diagnosis and preventive treatments' strategies of free radical related diseases of the newborn.

High-throughput plasma docetaxel quantification by liquid chromatography-tandem mass spectrometry.

BACKGROUND: The most valuable treatment option for breast, prostate and lung carcinomas is at present represented by a low dose of docetaxel, administered on a weekly basis. A better understanding of docetaxel pharmacokinetic and pharmacodynamic profiles could lead to an improvement in this dose regimen efficacy. METHODS: In this study a high-throughput method is described for the rapid quantification of docetaxel for large clinical pharmacology investigations. This analytical approach is based on an automatic on-line purification and enrichment technique followed by a measurement in tandem mass spectrometry through Multiple Reaction Monitoring. RESULTS: The assay was validated over a 0.15-1500 ng/mL range. Intra-day precision ranged from 1.9% to 6.4%, while the inter-day was between 7.6% and 11.2%. The mean deviation from the nominal value ranged from -0.5% to 5.6% for the intra-day, and from -0.4% to 3.1% for the inter-day assay. Clinical applicability was demonstrated by measuring plasma pharmacokinetics in patients receiving weekly 25-35 mg/m(2) of docetaxel. CONCLUSION: The proposed LC-MS/MS assay was found to have a better performance than previously reported methods in terms of sensitivity and sample preparation. It does not require any laborious pre-analytical manipulation and can be easily employed in large clinical pharmacology studies.

Serum steroid profiling by isotopic dilution-liquid chromatography-mass spectrometry: comparison with current immunoassays and reference intervals in healthy adults.

BACKGROUND: The simultaneous, rapid and reliable measurement of a wide steroid panel is a powerful tool to unravel physiological and pathological hormone status. Clinical laboratories are currently dominated by high-throughput immunoassays, but these methods lack specificity due to cross-reactivity and matrix interferences. We developed and validated an isotopic dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method for the simultaneous measurement of cortisol, corticosterone, 11deoxycortisol, androstenedione, deoxycorticosterone (DOC), testosterone, 17OHprogesterone, dehydroepiandrosterone (DHEA) and progesterone in serum, and compared it to routine immunoassays employed in our laboratory. We also established adult reference intervals in 416 healthy subjects. METHODS: 0.9 ml of serum were spiked with labelled internal standards (IS) and extracted on C18 cartridges. Eluate was injected into a two-dimensional LC-system, purified in a perfusion column and separated on a C8 column during a 21 min gradient run. Analytes were revealed by atmospheric pressure chemical ionization (APCI) followed by multiple reaction monitoring (MRM) analysis. RESULTS: Of the four immunoassays compared with the ID-LC-MS/MS method, only the results of ElecsysE170 for cortisol, testosterone in males and progesterone>1 ng/ml were in agreement with ID-LC-MS/MS. ElecsysE170 for testosterone in females and progesterone<1 ng/ml, Immulite2000 for androstenedione, DSL-9000 for DHEA and 17OHP Bridge for 17OHprogesterone, respectively, showed poor agreement. Reference intervals and steroid age and fertility related fluctuations were established. CONCLUSION: Our ID-LC-MS/MS method proved to be reliable and sensitive in revealing steroid circulating concentrations in adults and in highlighting the limits of routine immunoassays at low concentrations.

Fast liquid chromatography-tandem mass spectrometry method for routine assessment of irinotecan metabolic phenotype.

Irinotecan (CPT-11) is an anticancer drug with a complex in vivo metabolism widely used in the treatment of colon cancer. The assessment of the CPT-11 metabolic phenotype is an important clinical component for personalizing the administered dose. In this study, we report the development of a rapid and sensitive liquid chromatography-tandem mass spectroscopy method for the simultaneous quantitation of CPT-11 and its major metabolites generated by hydrolysis (SN-38, active metabolite) or through oxidative (NPC and APC) and glucuronidation (SN-38G) pathways. The method used a small volume of plasma and is based on simple protein plasma precipitation with two volumes of acetonitrile containing camptothecine as an internal standard. Analytes extracted in the supernatant fraction were converted to the lactone form and further separated by an acetonitrile gradient on a Kinetex C18 (50 × 2 mm) column in the presence of 0.01% formic acid. The quantitative measurement was performed by an API 4000/Qtrap operating in the triple-quadruple mode. The Multiple Reaction Monitoring transitions were: m/z 587→167 for CPT-11, m/z 393→349 for SN-38, m/z 619→393 for APC, m/z 519→393 for NPC, and m/z 569→393 for SN-38G. The mean overall recovery in plasma was in the range of 78% to 86% with a low matrix suppression effect (9.0% or less). The lower limit of quantitation was 0.2 ng/mL for NPC and SN-38 and 0.5 ng/mL for SN-38G, APC, and CPT-11. Linearity was checked up to 2000 ng/mL, whereas the intra- and interday accuracy and precision for both the parent drug and its metabolites was -3.7% to 13.8% and 3.7% to 13.1%, respectively. The method proved to be robust and suitable for therapeutic drug monitoring as well as for pharmacogenetic-pharmacokinetic correlation studies.

Liquid chromatography tandem mass spectrometry assay for fast and sensitive quantification of estrone-sulfate.

BACKGROUND: The circulating pool of estrone-sulfate is considered as a "reservoir" of slowly-metabolized estrogen that can be exploited for assessing overall individual estrogenicity. The aim of this study was to develop a rapid and sensitive liquid chromatography-tandem mass spectrometry assay for the determination of estrone-sulfate, suitable for routine clinical investigations. METHODS: The proposed assay is based on a simple protein precipitation procedure and on a fast measurement with a triple-quadrupole mass spectrometer operating in negative ion mode and in multiple reaction monitoring. The method was assessed for intra- and inter-day precision, accuracy, recovery, and clinical suitability. A comparison with available radioimmunoassay was also performed. RESULTS: The LC-MS/MS method is able to detect estrone-sulfate concentrations < or =1pg/mL and has a low limit of quantification of 7.8pg/mL. Intra- and inter-day precision and accuracy were less than 10.5% and 5.0% respectively. The recovery was in the range of 93%-110%. When compared with radioimmunoassay the method resulted more accurate and therefore more suitable for quantifying the estrone-sulfate in different clinical settings, including patients treated with aromatase inhibitors. CONCLUSIONS: The proposed LC-MS/MS method represents a convincing alternative to the immunoassay for a fast, cost-effective and reliable measurement of estrone-sulfate in routine clinical investigations and in large epidemiological studies. It may contribute in shedding a new light on the diagnostic value of estrone-sulfate in normal and pathological conditions.

Development of a method for the quantification of 1alpha,25(OH)2-vitamin D3 in serum by liquid chromatography tandem mass spectrometry without derivatization.

Quantification of 1alpha,25(OH)(2)-vitamin D(3) in serum is technically challenging because of its very low concentration. Even mass spectrometry faces a challenge due to the low sensitivity for this molecule, unless a specific derivatization is implemented. Therefore, there is still a need for a robust, simplified, sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantification of 1alpha,25(OH)(2)-vitamin D(3). Thanks to tandem mass- spectrometry associated to an on-line sample extraction and clean-up, sample preparation has been reduced to a simple protein precipitation step and derivatization has been avoided. Specimen volume has been kept to a minimum. The innovative aspects of the hereby-presented method are the use of stable lithium adducts and a highly sensitive tandem mass spectrometer. The achieved limit of quantification is at a level of 15 pg mL(-1), with a % CV of 5-15% at physiological concentration levels. The linearity is good and spiking experiments yielded a recovery of 87-102%. Comparison of selected samples with an established reference method, using an extensive purification procedure (high-performance liquid chromatography and radioimmunoassay), has shown a good correlation.

Determination of total homocysteine in plasma using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS).

A detailed protocol using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) is described for the determination of the total homocysteine (HCY) in plasma. Sample preparation is simple and relies on the complete reduction of any homocystine ((HCY)(2)) or protein-bound homocysteine, followed by the plasma protein precipitation. Any factor influencing the measurement is addressed. The protocol is robust and suitable for large-scale routine determinations.

New strategy for the screening of lysosomal storage disorders: the use of the online trapping-and-cleanup liquid chromatography/mass spectrometry.

The aim of this study was to set up a robust method suitable for large-scale studies (screening) with a minimized preparation process and with reduced running costs, for measuring five enzyme activities on dried blood spots by a new and simplified tandem mass spectrometry-based method. After incubation, all 5 reaction mixtures, carried out separately, were stopped, combined together, and centrifuged. The cleaning-up of the injected mixture was performed through a fast online trapping step preceding a liquid chromatography/tandem mass-spectrometry measurement. This method takes only 4 min as analysis run time and without any purification following the enzymatic reaction. We assessed the effectiveness of this approach in assaying the enzymatic activities on dried blood spots from 10 patients affected by "Pompe", 6 by "Gaucher", 12 by "Fabry", 3 by "Niemann-Pick" A/B, and 2 by "Krabbe" diseases. Reference values were established on 5000 healthy newborns and 300 healthy adults. All affected patients showed enzymatic activities below the normal range. In heterozygous carriers (18 for Fabry, 10 for Pompe, and 4 for Gaucher disease) the activities were slightly lower than in control subjects. The results show that the method set out in its simplicity, low costs, and low processes preparations can be fully applicable to a mass screening.

A liquid chromatography-tandem mass spectrometry method for the simultaneous determination of exemestane and its metabolite 17-dihydroexemestane in human plasma.

A simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantitation of exemestane (Exe) and its main metabolite 17-dihydroexemestane (DhExe) in human plasma. The analytes were extracted by protein precipitation with acetonitrile, containing stable 13C-labelled Exe (13C3-Exe) as internal standard, and measured by LC-MS/MS. The best chromatographic separation of the analytes from the interferences was achieved by using a Phenyl column operating under isocratic regime conditions. The total chromatographic runtime was 5.0 min and the elution of Exe and DhExe occurred at 2.5 min and 2.9 min, respectively. Quantitation was performed by employing the positive electrospray ionization (ESI) technique and multiple reaction monitoring mode (MRM). The monitored precursor to product-ion transitions for Exe, DhExe and 13C3-Exe internal standard were m/z 297.0 --> 120.8, m/z 299.1 --> 134.9 and m/z 300.0 --> 123.2, respectively. The lower limit of quantitation (LLOQ) was 0.1 ng/ml for DhExe and 0.2 ng/ml for Exe. The method was linear up to 36-51 ng/ml with r2 > or = 0.998. The intra- and inter-assay precision were < or = 7.7% and 5.1% for Exe and < or = 8.1 and 4.9% for DhExe while deviations from nominal values were in the 1.5-13.2% and - 9.0-5.8% ranges for Exe and DhExe, respectively. The analytical method resulted robust and suitable for pharmacokinetic monitoring of Exe and its main metabolite during adjuvant therapy in patients with breast cancer.

Setting up a 2D-LC/MS/MS method for the rapid quantitation of the prostanoid metabolites 6-oxo-PGF(1alpha) and TXB2 as markers for hemostasis assessment.

6-oxo-PGF(1alpha) and TXB(2) are the metabolites of the prostanglandin PGI(2) and of the thromboxane TXA(2), respectively. PGI(2) and TXA(2) are arachidonic acid-derived compounds which regulate the blood hemostasis. Their quick metabolism leads to the 6-oxo-PGF(1alpha) and TXB(2) metabolites in plasma. In order to study on a large base the external factors influencing the hemostatic conditions, there is a need for a fast and reliable assay for quantitating these metabolites. Some methods have been published for the analysis of the arachidonic acid-derived compounds and some are dealing with mass spectrometry but nonspecifically centered on these specific compounds with a fast and cheap protocol, amenable for large-scale studies. Here we describe an analytical strategy that incorporates a two-dimensional chromatography running coupled to tandem mass spectrometry that minimizes the sample preparation and addresses the presence of the TXB(2) anomers for a robust quantitation measurement. After a protein precipitation, 100 microl of the supernatant (corresponding to 50 microl of the original plasma) was injected in a two-dimensional chromatographic system which operates an on-line clean-up and a subsequent chromatographic separation of the targeted analytes with a limit of quantitation (LOQ) of 22 pg/ml for 6-oxo-PGF(1alpha), and and a LOQ of 25 pg/ml for TXB(2).

Rapid and sensitive analysis of vincristine in human plasma using on-line extraction combined with liquid chromatography/tandem mass spectrometry.

A simple and rapid method has been developed and validated for the quantitation of vincristine in human plasma by liquid chromatography/tandem mass spectrometry (LC/MS/MS) with atmospheric pressure chemical ionization using on-line solid-phase extraction. The method uses vinblastine as internal standard and the sample preparation is limited just to a plasma protein precipitation step. Further sample clean-up is carried out on-line through a perfusion column preceding an analytical phenyl LC column, the latter directly connected to the mass spectrometer. Quantitation is performed in multiple reaction monitoring mode using the transitions of m/z 825.3 --> 765.3 and 811.3 --> 751.3 for vincristine and vinblastine respectively. The assay was linear (r2 > or =0.99) in a concentration range from 0.1 to 500 ng/mL. Carry-over, measured on the experimental set-up, was less than 0.04%. Recovery for vincristine and the internal standard was within 90-95%. The intra-day and inter-day assay precision ranged from 1.2% to 6.8% RSD while mean percentage deviation from nominal value ranged from 0.01% to 6.1%. The proposed assay was found suitable for pharmacokinetics investigations and clinical therapeutic drug monitoring especially in pediatric cancer patients.