Microscale high-performance liquid chromatography–electrospray tandem mass spectrometry assay for cyclosporin A in blood

To facilitate quantitative analysis of cyclosporin A in low volume blood samples we developed a sensitive and specific microscale reversed-phase HPLC–electrospray tandem mass spectrometry assay. Blood samples (100 μl) were prepared by acetonitrile precipitation and C₁₈ solid-phase extraction. Detection was by multiple-reactant monitoring. The method was linear over the range 5–1000 μg/l (r≥0.997) with accuracy between 95.4 and 102.0% over this range. Total imprecision was 11.1% at 10 μg/l and 2.8% at 800 μg/l. Absolute recovery of cyclosporin A and internal standard was 72.5 and 73.3%, respectively. When this method was evaluated against a conventional HPLC with UV detection, in patient samples, they were interchangeable (y=0.988x+10.0, r=0.996). This HPLC–ESI-MS–MS method will be applicable to therapeutic monitoring in paediatric transplant patients and multiple point pharmacokinetic studies in animals and humans.     

A fast and sensitive HPLC–MS/MS analysis and preliminary pharmacokinetic characterization of ergone in rats

A fast and sensitive HPLC–APCI-MS/MS method was developed for the determination of ergosta-4,6,8(14),22-tetraen-3-one (ergone) in rat plasma. The plasma sample containing ergone and ergosterol (internal standard) were simply treated with acetone to precipitate and remove proteins and the isolated supernatants were directly injected into the HPLC–APCI-MS/MS system. Chromatographic separation was performed on a 1.8 μm Zorbax SB-C18 column (100 mm × 3.0 mm) with a 97:3 (v/v) mixed solution of methanol and 0.1% aqueous formic acid being used as mobile phase. Quantification was performed by multiple selected reactions monitoring (MRM) of the transitions with (m/z)⁺ 393–268 for ergone and (m/z)⁺ 379–69 for the IS. The method was validated in the concentration range of 5–1600 ng/mL for ergone. The precision of the assay (RSD%) was less than 10.5% at all concentrations levels within the tested range and adequate accuracy, and the limit of detection was 1.5 ng/mL. The absolute recoveries of both ergone and ergosterol from the plasma were more than 95%. The developed method has been successfully applied to the pharmacokinetic study of the drug in SD rats.     

Method for measurement of the quaternary amine compounds paraquat and diquat in human urine using high-performance liquid chromatography–tandem mass spectrometry

We have developed a highly selective and sensitive analytical method to quantify paraquat and diquat by use of high-performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS). The sample preparation includes solid phase extraction that uses weak cation exchange cartridges. These highly charged dual quaternary amines were not retained by standard reversed phase columns, but they could be adequately separated through HPLC with a HILIC column. The detection was carried out with a triple quadrupole mass spectrometer with an electrospray ionization probe in positive ion mode in multiple reaction monitoring. Repeated analysis in human urine samples spiked with low (5 ng/ml), medium (15 ng/ml), and high (30 ng/ml) concentrations of the analytes yielded relative standard deviations of less than 9%. The extraction efficiencies ranged from 77.7% to 94.2%. The limits of detection were in the range of 1 ng/ml.     

Automated mass spectrometric analysis of urinary free catecholamines using on-line solid phase extraction

Analysis of catecholamines (epinephrine, norepinephrine and dopamine) in plasma and urine is used for diagnosis and treatment of catecholamine-producing tumors. Current analytical techniques for catecholamine quantification are laborious, time-consuming and technically demanding. Our aim was to develop an automated on-line solid phase extraction method coupled to high performance liquid chromatography–tandem mass spectrometry (XLC–MS/MS) for the quantification of free catecholamines in urine. Five microlitre urine equivalent was pre-purified by automated on-line solid phase extraction, using phenylboronic acid complexation. Reversed phase (pentafluorophenylpropyl column) chromatography was applied. Mass spectrometric detection was operated in multiple reaction monitoring mode using a quadrupole tandem mass spectrometer with positive electrospray ionization. Urinary reference intervals were set in 24-h urine collections of 120 healthy subjects. XLC–MS/MS was compared with liquid chromatography with electrochemical detection (HPLC–ECD). Total run-time was 14 min. Intra- and inter-assay analytical variations were <10%. Linearity was excellent (R² > 0.99). Quantification limits were 1.47 nmol/L, 15.8 nmol/L and 11.7 nmol/L for epinephrine, norepinephrine and dopamine, respectively. XLC–MS/MS correlated well with HPLC–ECD (correlation coefficient >0.98). Reference intervals were 1–10 μmol/mol, 10–50 μmol/mol and 60–225 μmol/mol creatinine for epinephrine, norepinephrine and dopamine, respectively. Advantages of the XLC–MS/MS catecholamine method include its high analytical performance by selective PBA affinity and high specificity and sensitivity by unique MS/MS fragmentation.     

Determination of free and glucuronidated kaempferol in rat plasma by LC–MS/MS: Application to pharmacokinetic study

Flavanoid kaempferol is mainly present as glucuronides and sulfates in rat plasma, and small amounts of the intact aglycone are also detected. In the this study, a rapid, specific and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry method (HPLC–MS/MS) was developed and validated for determination of kaempferol and its major metabolite glucuronidated kaempferol in rat plasma. A liquid–liquid extraction with acetic ether was involved for the extraction of kaempferol and internal standard. Analytes were separated on a C18 column (150 mm × 2.1 mm, 4.5 μm, Waters Corp.) with isocratic elution at a flow-rate of 0.3 ml min⁻¹. The mobile phase was consisted of 0.5% formic acid and acetonitrile (50:50, v/v). The Quattro Premier HPLC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. The method was validated according to the FDA guidelines for validation of bioanalytical method. The validated method was successfully applied to the study of the pharmacokinetics in rats after oral administration of kaempferol with different doses.     

Identification of quercetin glucuronides in human plasma by high-performance liquid chromatography–tandem mass spectrometry

After intake of food or herbal medicinal products containing quercetin glycosides, the systemic availability of the genuine glycoside, as well as the systemic occurrence of the aglycone or conjugates of this polyphenol has been a matter of dispute. Consequently, we designed this study to develop a reliable method for determination of quercetin and its metabolites. Following consumption of fried onions five different glucuronides of quercetin could be identified in human plasma samples by means of HPLC–UV–MS/MS. Selective determination of the target compounds was achieved by simultaneous UV (254 nm) and MS/MS detection with selected reaction monitoring experiments using positive mode electrospray ionisation. In contrast, neither the free flavonol nor the genuine glycoside could be detected in plasma. Identification of the quercetin glucuronides detected in vivo was confirmed by comparison with authentic reference compounds synthesised enzymatically using glucuronyl transferase from rabbit liver.     

Analysis of global DNA methylation by hydrophilic interaction ultra high-pressure liquid chromatography tandem mass spectrometry

We developed and validated a rapid, sensitive, and specific liquid chromatography tandem mass spectrometry (LC–MS/MS) method for determination of global DNA methylation in tissue. DNA was extracted by phenol–chloroform, hydrolyzed using 88% formic acid at 140 °C, spiked with cytosine-2,4-¹³C¹⁵N₂ as internal standard, evaporated under nitrogen, reconstituted in methanol, and analyzed by LC–MS/MS in multiple reaction monitoring mode to reflect the global DNA methylation of the tissue. The method was linear throughout the range of clinical interest and had good sensitivity, with a limit of quantification of 0.5 pg for both cytosine (Cyt) and 5-methylcytosine (5mCyt). The linear range of calibration curve was 1–50 and 1–100 ng/ml for 5mCyt and Cyt, respectively, with a correlation coefficient higher than 0.99. The relative standard deviation (RSD) was 0.70–4.09% and 0.60–4.81% for Cyt and 5mCyt, respectively. The intraday precision expressed as RSD ranged from 1.86% to 4.67%, whereas the interday values ranged from 3.72% to 4.68%. The recovery of the method varied from 86.52% to 105.14%. This yielded a simple and reliable LC–MS/MS assay for detection of Cyt and 5mCyt, thereby enabling the evaluation of global DNA methylation.     

Comparison of high-performance liquid chromatography with electrochemical detection and gas chromatography—mass fragmentography for the assay of salsolinol, dopamine and dopamine metabolites in food and beverage samples

High-performance liquid chromatography with electrochemical detection (HPLC—ED) and combined gas chromatograph—mass spectrometry in the single-ion monitoring mode (GC—MS-SIM) have been used for the determination of salsolinol, dopamine, 3,4-dihydroxyphenylacetic acid, 3,4-dihydorxyphenylethanol and norepinephrine in a selection of food and beverage samples. The unique specificity of the SIM mode allows a simple one-step extraction to be used even for complex sample matrices. We have been able to demonstrate the quantitative and qualitative advantages offered by GC—MS over HPLC—ED by direct comparison of the chromatographic data obtained. We demostrate that the specificity of SIM and the benefits offered by the incorporation of deuterated internal standards make GC—MS-SIM the method of choice for valid identification and precise quantitation of salsolinol, dopamine and dopamine metabolites in a complex sample matrix.     

LC–MS/MS coupled with immunoaffinity extraction for determination of estrone, 17β-estradiol and estrone 3-sulfate in human plasma

Determination of estrogens in plasma is important in evaluation of effects of some anticancer drugs, such as aromatase inhibitors. However, as reported previously, high performance liquid chromatography–radio immunoassay (HPLC–RIA) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) with chemical derivatization require complicated sample preparation. In this study, a highly sensitive and simple method for determination of estrone (E1), 17β-estradiol (E2) and estrone 3-sulfate (E1S) in human plasma has been developed. Following diethylether extraction from plasma, analytes were purified by immunosorbents and then determined by LC–MS/MS using electrospray ionization (ESI). Immunosorbents were prepared by immobilization of specific antibodies raised against each analyte onto solid support. Use of selective immunosorbents in sample preparation removed interference in plasma samples that would cause ionization suppression, and markedly improved the sensitivity of LC–MS/MS for these analytes, without derivatization. Calibration curves of each analyte showed good linearity and reproducibility over the range of 0.05–50 pg/injection for E1, 0.2–50 pg/injection for E2 and 0.05–300 pg/injection for E1S, respectively. The mean values of lower limits of quantification (LLOQ) in human plasma corrected by recovery of deuterated estrogens (internal standard, I.S.) were 0.1892 pg/mL for E1, 0.7064 pg/mL for E2 and 0.3333 pg/mL for E1S, respectively. These LLOQ values were comparable to those previous reported using HPLC–RIA and LC–MS/MS. Using this method, the normal levels of three estrogens in healthy female plasma (n = 5) were determined. The mean values of E1, E2 and E1S were 38.0 pg/mL (range 24.8–53.0), 34.3 pg/mL (22.6–46.6) and 786 pg/mL (163–2080), respectively. The immunoaffinity LC–MS/MS described here allows sensitive and accurate quantification of E1, E2 and E1S without laborious sample preparation.     

A new peptide substrate for enhanced botulinum neurotoxin type B detection by endopeptidase–liquid chromatography–tandem mass spectrometry/multiple reaction monitoring assay

Botulinum neurotoxins (BoNTs) are the most toxic proteins in nature. Rapid and sensitive detection of BoNTs is achieved by the endopeptidase–mass spectrometry (Endopep–MS) assay. In this assay, BoNT cleaves a specific peptide substrate and the cleaved products are analyzed by MS. Here we describe the design of a new peptide substrate for improved detection of BoNT type B (BoNT/B) in the Endopep–MS assay. Our strategy was based on reported BoNT/B–substrate interactions integrated with analysis method efficiency considerations. Incorporation of the new peptide led to a 5-fold increased sensitivity of the assay both in buffer and in a clinically relevant human spiked serum.     

Synthesis of haptens and development of an indirect enzyme-linked immunosorbent assay for tris(2,3-dibromopropyl) isocyanurate

A competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed for detection of tris(2,3-dibromopropyl) isocyanurate (TBC). Polyclonal antibodies against TBC were raised from synthesized haptens and then screened against various coating antigens. After optimization of the immunoassay conditions, the linear range and IC₅₀ value of the assay were 0.30–100 and 5.17 μg/L, respectively, with little cross-reactivity (?2%). Recovery of various samples (water, serum, soil) was tested and the values ranged from 68% to 110%. This ciELISA was also applied to determine TBC in the riverside soil of the Liuyang River, and the results were compared with the data obtained by UHPLC–MS/MS. The experimental assay results confirmed that this proposed immunoassay is a specific, sensitive, and reliable method for determination and monitoring of TBC.     

Determination of cyclophosphamide and its metabolites in human plasma by high-performance liquid chromatography–mass spectrometry

A sensitive HPLC–MS method was developed for the simultaneous determination of cyclophosphamide and its metabolites 4-hydroxycyclophosphamide (aldocyclophosphamide), 4-ketocyclophosphamide, caboxyphosphamide and 3-dechloroethylifosfamide in human plasma. 4-Hydroxycyclophosphamide was converted with methylhydroxylamine to the stable methyloxime form. We used a solid-phase extraction with C₁₈ cartridges followed by HPLC–MS with the single mass spectrometer SSQ 7000 of Finnigan. The limits of detection were 15 ng/ml for cyclophosphamide, 3-dechloroethylifosfamide and ketocyclophosphamide in each case and 30 ng/ml for carboxyphosphamide and 4-hydroxycyclophosphamide, respectively. First results of pharmacokinetics are shown.     

Urinary analysis of 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2′-deoxyguanosine by isotope-dilution LC-MS/MS with automated solid-phase extraction: Study of 8-oxo-7,8-dihydroguanine stability

A highly sensitive quantitative method based on LC-MS/MS was developed to simultaneously and directly measure 8-oxo-7,8-dihydroguanine (8-oxoGua) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) in urine. It was found that 8-oxoGua could be artifactually generated from 8-oxodGuo during the ionization process by both in-source thermolysis and collisionally induced dissociation. Our method applied a two-stage wash procedure in the online solid-phase extraction system that not only eliminated ion suppression but also prevented artifactual interference with 8-oxoGua by 8-oxodGuo by eluting the analytes individually. With the use of isotope internal standards, the detection limits of 8-oxoGua and 8-oxodGuo were estimated to be 30 and 3.5 fmol, respectively. The 8-oxoGua stability under common storage conditions was first investigated. Dissolved 8-oxoGua in NaOH (pH 12) was quite fragile and stable for only < 1 day at room temperature. When pH and temperature were reduced, the 8-oxoGua stability at ? 20°C was significantly increased to ～ 87 days in water (pH ～ 7) and ～ 112 days when diluted in 5% methanol. This method was further applied to measure urinary samples of healthy subjects. A molar ratio of 8-oxoGua to 8-oxodGuo of ～ 4.6 was found, supporting the hypothesis that oxidatively damaged DNA is primarily repaired by the base excision repair pathway.     

A fast and sensitive HPLC-MS/MS analysis and preliminary pharmacokinetic characterization of cudratricusxanthone B in rats

A method for the quantitative analysis of cudratricusxanthone B (CXB) in rat plasma by high performance liquid chromatography–electrospray ionization-tandem mass spectrometry (HPLC–ESI-MS/MS) has been developed and validated. The method involved liquid–liquid extraction from plasma, simple chromatographic conditions on a Venusil XBP-PH C₁₈ column with the mobile phase of 0.5% formic acid in methanol, and mass spectrometric detection using an API-3000 instrument. Multiple reaction monitoring (MRM) mode was used to monitor precursor/product ion transitions of m/z 397.1/285.0 for CXB and m/z 381.6/269.2 for the internal standard (I.S.) cudraxanthone H. The standard curves were linear over the concentration range of 1–500 ng/mL for CXB in rat plasma. The intra- and inter-batch accuracy for CXB at four concentrations was 89.4–99.5% and 89.4–100.8%, respectively. The RSDs were less than 7.92%. The lower limit of quantification for CXB was 1.0 ng/mL using 100 μL of plasma. The average extraction recoveries of CXB ranged from 80.1 to 95.4% at the concentrations of 2, 50 and 500 ng/mL, respectively. This method was successfully applied to the pharmacokinetic study after an intravenous administration of CXB in male Sprague–Dawley (SD) rats.     

DNA strand breaks and base modifications induced by cholesterol hydroperoxides

Abstract

Cholesterol (Ch) can be oxidized by reactive oxygen species, forming oxidized products such as Ch hydroperoxides (ChOOH). These hydroperoxides can disseminate the peroxidative stress to other cell compartments. In this work, the ability of ChOOH to induce strand breaks and/or base modifications in a plasmid DNA model was evaluated. In addition, HPLC/MS/MS analyses were performed to investigate the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) after the incubation of 2′-deoxyguanosine (dGuo) with ChOOH and Cu²⁺. In the presence of copper ions, ChOOH induced DNA strand breaks in time and concentration-dependent manners. Purine and pyrimidine base modifications were also observed, as assessed respectively by the treatment with Fpg and Endo III repair enzymes. The detection of 8-oxodGuo by HPLC/MS/MS is in agreement with the dGuo oxidation in plasmid DNA. ChOOH-derived DNA damage adds further support to the role of lipid peroxidation in inducing DNA modifications and mutation.     

An improved on-line solid phase extraction coupled HPLC–MS/MS system for quantification of Sifuvirtide in human plasma

An improved liquid chromatographic method with on-line solid phase extraction (SPE) and tandem mass spectrometric detection was optimised for quantification of the anti-HIV peptide Sifuvirtide in human plasma. The SPE sorbents, loading buffer composition and other aspects of the on-line SPE column were investigated in detail for efficiently extracting the interesting peptides and simultaneously discarding the large amount of proteins. The gradient elution program was optimised on the analysis column to decrease the matrix effect and obtain excellent selectivity. The multiple charge ion at m/z 946.4 of Sifuvirtide was quantified by a linear ion trap mass spectrometer, operating in the positive mode, and selective reaction monitoring (SRM) acquisition. Method validation results demonstrated that the linear calibration curve covered a range of 6.1–6250 ng/mL, and the correlation coefficients (r²) were above 0.992. The lower limit of detection (LLOD) with a signal-to-noise (S/N) ratio higher than 10 was 6.1 ng/mL. The accuracy ranged from −7.6 to 10.6%, and the intra- and inter-batch precisions were less than 8.7% and 5.5%, respectively. Finally, more than nine hundred of samples from a clinical trial was completely analyzed using this on-line SPE coupled HPLC–MS/MS system in one single week, due to the rapid run-time of individual sample (6.5 min).     

Quantification of mycophenolate mofetil in human skin extracts using high-performance liquid chromatography–electrospray mass spectrometry

A sensitive and selective method for the quantification of mycophenolate mofetil and its active metabolite mycophenolic acid in different human skin layers after dermal administration is presented. The skin layers were separated after in vitro penetration experiments and a methanolic extraction was performed. Positive ion electrospray HPLC–MS in selected ion monitoring mode was used to quantify the substances after isocratic separation by a C₁₈ analytical column. The minimum detectable concentrations were 850 pg/ml for MMF and 1 ng/ml for MPA. The peak areas depended linearly on the concentration of both drugs over the range of 25–1000 ng/ml (r²≥0.996) with accuracy ≤9.8% and precision ≤13.2%. Total imprecision at quantification limits was 15.2% at 10 ng/ml and 16.3% at 1500 ng/ml for MMF and 15.1% at 21.0 ng/ml and 17.5% at 1300 ng/ml for MPA. This HPLC–MS method will be applicable to the profiling of MMF amounts in skin and its conversion to MPA after application of different formulations.     

Effects of arsenic exposure among semiconductor workers: a cautionary note on urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine

Arsenic is a notorious environmental toxicant and was found to cause oxidative stress in cultured cells and animals. However, little work has been done in human studies, especially for the population occupationally exposed to arsenic. In order to investigate the effect of occupational exposure to arsenic in oxidative stress, we measured urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) from 90 semiconductor workers including 50 exposed and 40 nonexposed subjects. A highly sensitive and specific isotope dilution LC-MS/MS method was used for quantification of 8-oxodGuo. The levels of inorganic arsenic (iAs3+, iAs5+), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) in urine were determined by high-performance liquid chromatography-flow injection atomic absorption spectrometry (HPLC-FIAAS). Results showed that the mean urinary concentrations of total arsenic and 8-oxodGuo were significantly higher for exposed workers compared with the nonexposed workers. In addition, elevated urinary 8-oxodGuo concentrations of exposed workers were correlated with urinary levels of MMA (r = 0.44, P < 0.005) and the extent of primary methylation (the ratio of MMA to inorganic arsenic) (r = 0.40, P < 0.005). These findings suggested that occupational exposure to arsenic could result in the induction of oxidative stress. The presence and/or formation of MMA could play an important role in arsenic-involved injuries.     

Liquid chromatographic determination of myocardial interstitial epinephrine

This study describes a high-performance liquid chromatographic method with electrochemical detection (HPLC–ED) for monitoring of epinephrine (Epi) in the myocardial interstitial space. The in vitro detection limit for Epi was 200 fg in a 50-μl injection. Using a cardiac dialysis technique, 60-μl dialysates were sampled from the myocardial interstitial space (6-min fractions). After an alumina procedure, the dialysate Epi concentration was measured using the HPLC–ED system. Although the basal Epi concentration was undetectable, local administration of desipramine increased Epi concentration of the dialysate to 38.1±18.5 pg/ml. This system affords a new possibility for estimating myocardial interstitial Epi level.     

DNA strand breaks and base modifications induced by cholesterol hydroperoxides

Cholesterol (Ch) can be oxidized by reactive oxygen species, forming oxidized products such as Ch hydroperoxides (ChOOH). These hydroperoxides can disseminate the peroxidative stress to other cell compartments. In this work, the ability of ChOOH to induce strand breaks and/or base modifications in a plasmid DNA model was evaluated. In addition, HPLC/MS/MS analyses were performed to investigate the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) after the incubation of 2'-deoxyguanosine (dGuo) with ChOOH and Cu(2+). In the presence of copper ions, ChOOH induced DNA strand breaks in time and concentration-dependent manners. Purine and pyrimidine base modifications were also observed, as assessed respectively by the treatment with Fpg and Endo III repair enzymes. The detection of 8-oxodGuo by HPLC/MS/MS is in agreement with the dGuo oxidation in plasmid DNA. ChOOH-derived DNA damage adds further support to the role of lipid peroxidation in inducing DNA modifications and mutation.