Quantitation of lower levels of the DNA adduct of thymidylyl(3'-5')thymidine methyl phosphotriester by liquid chromatography/negative atmospheric pressure chemical ionization tandem mass spectrometry

A sensitive method has been developed for the direct quantitation of the methyl phosphotriester DNA adduct of thymidyl(3'-5')thymidine (dTp(Me)dT) from enzymatic DNA hydrolysates prepared from cultured cells treated with low levels of N-methyl-N-nitrosourea (MNU) and methyl methane sulfonate (MMS), by rapid and selective liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry (LC/APCI-MS/MS). The lower limit of quantitation was 0.1 ng/mL (6.4 adducts per 10(8) nucleotides). Linearity of the calibration curve was greater than 0.999 from 0.1 to 50 ng/mL. Intra-day precision for three levels of quality control samples ranged from 4.27 to 15.62%. Interday precision ranged from 2.46 to 11.95%. Using this method, the levels of dTp(Me)dT in DNA enzymatic hydrolysates obtained from a series of incubations of mouse lymphoma cells with low doses of MNU (50 microM) were quantified.     

Differential adduction of proteins vs. deoxynucleosides by methyl methanesulfonate and 1-methyl-1-nitrosourea in vitro

The reactions of two model mutagenic and carcinogenic alkylating agents, N-methyl-N-nitrosourea (MNU) and methyl methanesulfonate (MMS), with proteins and deoxynucleosides in vitro, were investigated. The protein work used an approach involving trypsin digestion and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). This technique permitted identification of the specific location of protein adduction by both MNU and MMS with commercial apomyoglobin and human hemoglobin, under physiological conditions. MNU treatment resulted in predominantly carbamoylation adducts on the proteins, but in contrast only methylated protein adducts were found following treatment with MMS. Further analyses, using TurboSequest, and the Scoring Algorithm for Spectral Analysis (SALSA), revealed that MNU carbamoylation was specific for modification of either the N-terminal valine or the free amino group in lysine residues of apomyglobin and human hemoglobin. However, MMS methylation modified the N-terminal valine and histidine residues of the proteins. Despite their clear differences in protein modifications, MNU and MMS formed qualitatively the same methylated deoxynucleoside adduct profiles with all four deoxynucleosides in vitro under physiological conditions. In light of their different biological potencies, where MMS is considered a 'super clastogen' while MNU is a 'super mutagen', these differences in reaction products with proteins vs. deoxynucleosides may indicate that these two model alkylating agents work via different mechanisms to produce their mutagenic and carcinogenic effects.     

Quantitation of DNA adduct of thymidylyl(3'-5')thymidine methyl phosphotriester by liquid chromatography/negative electrospray tandem mass spectrometry

A rapid and selective method based on liquid chromatography/electrospray tandem mass spectrometry (LC/ESI-MS/MS) has been developed for the direct quantitation of a methyl phosphotriester DNA adduct, thymidyl (3'-5') thymidine [dTp(Me)dT] from enzymatic hydrolysates of DNA (either in vitro DNA or in cell culture) treated with MNU (N-methyl-N-nitrosourea) or MMS (methyl methane sulfonate). The lower limit of quantitation was 2 ng/mL. Linearity of the calibration curve was greater than 0.999 from 2 to 1000 ng/mL. Intraday precision for four levels of quality controls ranged from 2.8 to 20.1%, and interday precision ranged from 2.9 to 5.6%. This method was used to quantify the levels of dTp(Me)dT in enzymatic hydrolysates of DNA obtained from a series of incubations of salmon testis DNA or mouse lymphoma cells with either MNU or MMS.     

Extraction of clenbuterol from calf urine using a molecularly imprinted polymer followed by quantitation by high-performance liquid chromatography with UV detection

A method for the extraction of clenbuterol from calf urine samples using a molecularly imprinted polymer (MIP) has been developed. The aim was that the final extracts from the MIP should allow quantitation of clenbuterol down to 0.5 ng/mL urine using HPLC with UV detection. The MIP was produced using brombuterol as a template and the selectivity of the MIP, for clenbuterol, was tested against a non-imprinted polymer (produced without template) and was found to be high. After loading of 5 mL diluted centrifuged urine, selective binding was established in acetonitrile-acetic acid (98:2). For further elution of interferences, 0.5 M ammonium acetate buffer pH 5 and 70% acetonitrile in water was used. Clenbuterol was eluted using 1% trifluoroacetic acid in methanol, which was evaporated and reconstituted in buffer. Results from the HPLC analyses showed that the extraction of clenbuterol using MIP is linear in the range 0.5-100 ng/mL with good precision (4.3% for 0.6 ng/mL and 2.1% for 6.0 ng/mL) and accuracy (96.7% for 0.6 ng/mL and 96.7% for 6.0 ng/mL). The recoveries were 75%. The results show that the method offers a selectivity and sensitivity that make the quantitation of 0.5 ng clenbuterol/mL urine by HPLC-UV possible and a competitive alternative to state-of-the-art routine analytical methods.     

Development of a validated method for the simultaneous determination of amphetamine,methamphetamine and methylenedioxyamphetamines (MDA,MDMA, MDEA) in serum by GC-MS after derivatisation with perfluorooctanoyl chloride

A rapid and simple method for the simultaneous detection and quantitation of amphetamine, methamphetamine, methylenedioxyamphetamine (MDA), methylenedioxymethamphetamine (MDMA) and methylenedioxyethylamphetamine (MDEA) in human serum was developed and fully validated. Serum samples were extracted with cyclohexane, derivatised with perfluorooctanoyl chloride without prior evaporation of the solvent and analysed with gas chromatography-mass spectrometry (GC-MS) in the selected ion monitoring mode (SIM). For quantitation, deuterated analogues were used as the internal standards. The limit of detection (LOD) and lower limit of quantitation (LLOQ), bias and within-day and between-day precision were determined. LODs calculated as the average of the six calibration curves were below 5 ng/mL for all of the measured compounds; LLOQs obtained in the same manner were below 20 ng/mL, with the exception of MDA (24.1 ng/mL). The coefficients of variation were below 7% within series, 10% or less between series and the bias was below 8% for all compounds. The calibration curves were linear between the lower limits of quantitation and 800 ng/mL. Presented in part at 12. Jahrestagung, Deutsche Gesellschaft für Rechtsmedizin, May 2003, Kiel, Germany.     

芥子气中毒人员尿液标记物谱分析及其临床诊断应用

该研究基于暴露生物标记物溯源思路,应用于解决芥子气(SM)临床早期诊断、溯源确证难题.建立了芥子气中毒患者尿液中7 种游离代谢产物的两步固相萃取/超高效液相色谱-串联质谱(SPE/UPLC-MS/MS)同时定量方法,检出限为5 pg/mL～1 ng/mL,定量下限为10 pg/mL～5 ng/mL;结合前期建立的4 种...     

Immuno‐capture as ultimate sample cleanup in LC‐MS/MS determination of the early stage biomarker ProGRP

This paper presents a selective and efficient sample preparation procedure for NLLGLIEAK, signature peptide for the small cell lung cancer (SCLC) biomarker ProGRP, in human serum. The procedure is based on immuno‐capture of ProGRP in 96‐wells microtiter plates coated with the mAb E146. After immuno‐capture and thorough rinse, trypsin was added for in‐well digestion. Subsequently the signature peptide was enriched by SPE and determined by LC‐MS/MS. Various steps in the procedure were optimized to achieve a low LOD such as dilution of sample, tryptic digestion, and SPE cleanup and peptide enrichment conditions. A single quadropole MS was used during optimization of the method. A triple quadropole MS was used in the method evaluation in order to improve sensitivity. The evaluation showed good repeatability (RSD, 11.9–17.5%), accuracy (3.0–6.6%), and linearity (r² = 0.995) in the tested range (0.5–50 ng/mL). LOD and LOQ were in the pg/mL area (0.20 and 0.33 ng/mL, respectively), enabling the determination of clinically relevant concentrations. The method was applied to two patient samples and showed good agreement with an established immunological reference method. The final method was compared to a previous published LC‐MS method for the determination of ProGRP in serum based on protein precipitation and online sample cleanup. Both showed acceptable method performance, however, the immuno‐capture LC‐MS method was superior with respect to sensitivity. This illustrates the large potential of immuno‐capture sample preparation prior to LC‐MS in protein biomarker quantification.     

Quantitation of the large polypeptide glucagon by protein precipitation and LC/MS

We present a method for the quantitation of glucagon from rat plasma by protein precipitation and LC/MS. No internal standard was used, as a labeled standard was not available and similar peptides did not show comparable extraction characteristics to glucagon. The LC system included a Keystone C18, 300 A pore size column; a linear gradient was used with a mobile phase consisting of water and acetonitrile, each with 0.2% acetic acid and 0.02% trifluoroacetic acid. Glucagon was detected with the mass spectrometer in positive ion mode monitoring the 4+ charge state at m/z 871.7. The method had an approximated limit of detection of 1 ng/mL. The lower limit of quantitation (LLOQ) was 25 ng/mL (7.2 fmol/mL), which could be reduced with an appropriate internal standard. External calibration was used and calibration curves were found to be linear over the range from 25 to 1000 ng/mL (7.2 to 290 fmol/mL). The method showed a high degree of precision and accuracy both within and between runs at four validation points, including the LLOQ.     

Analytical determination of specific 4,4′-methylene diphenyl diisocyanate hemoglobin adducts in human blood

4,4′-Methylene diphenyl diisocyanate (MDI) is one of the most important isocyanates in the industrial production of polyurethane and other MDI-based synthetics. Because of its high reactivity, it is known as a sensitizing agent, caused by protein adducts. Analysis of MDI is routinely done by determination of the nonspecific 4,4′-methylenedianiline as a marker for MDI exposure in urine and blood. Since several publications have reported specific adducts of MDI and albumin or hemoglobin, more information about their existence in humans is necessary. Specific adducts of MDI and hemoglobin were only reported in rats after high-dose MDI inhalation. The aim of this investigation was to detect the hemoglobin adduct 5-isopropyl-3-[4-(4-aminobenzyl)phenyl]hydantoin (ABP-Val-Hyd) in human blood for the first time. We found values up to 5.2 ng ABP-Val-Hyd/g globin (16 pmol/g) in blood samples of workers exposed to MDI. Because there was no information available about possible amounts of this specific MDI marker, the analytical method focused on optimal sensitivity and selectivity. Using gas chromatography–high-resolution mass spectrometry with negative chemical ionization, we achieved a detection limit of 0.02 ng ABP-Val-Hyd/g globin (0.062 pmol/g). The robustness of the method was confirmed by relative standard deviations between 3.0 and 9.8 %. Combined with a linear detection range up to 10 ng ABP-Val-Hyd/g globin (31 pmol/g), the enhanced precision parameter demonstrates that the method described is optimized for screening studies of the human population.     

Quantitative determination of the polypeptide motilin in rat plasma by externally calibrated liquid chromatography/electrospray ionization mass spectrometry

We present a method for the quantitation of motilin from rat plasma by protein precipitation and liquid chromatography/mass spectrometry (LC/MS). Using external calibration, the method was linear over the concentration range 10-1000 ng/mL with an initial sample volume of 150 microL. The LC system included a C(18) column with a 300 A pore size. A linear gradient was used with a mobile phase consisting of water and acetonitrile, each with 0.2% acetic acid and 0.02% trifluoroacetic acid. Motilin was detected with the mass spectrometer in positive ion mode monitoring the 4+ charge state at m/z 675.5. The approximated limit of detection was less than 1 ng/mL and the lower limit of quantitation (LLOQ) was 10 ng/mL. The method showed a high degree of precision and accuracy both within and between runs at five validation points, including the LLOQ.     

On-line extraction coupled with liquid chromatography tandem mass spectrometry for quantitation of pravastatin and its metabolite in human serum

A high-throughput bioanalytical method for simultaneous quantitation of pravastatin and its metabolite (M1) in human serum was developed and validated using on-line extraction following liquid chromatography tandem mass spectrometry (LC-MS/MS). The on-line extraction was accomplished by the direct injection of a 50 microL serum sample, mixed 4:1 with an aqueous internal standard solution, into one of the extraction columns with aqueous 1 mm formic acid at flow rate of 3 mL/min. The separation and analysis were achieved by back-eluting the analytes from the extraction column and the analytical column to the mass spectrometer with an isocratic mobile phase consisting of 62% aqueous 1 mm formic acid and 38% acetonitrile at a flow rate of 0.8 mL/min. The second extraction column was being equilibrated while the first column was being used for analysis, and vice versa. The standard curve range was 0.500-100 ng/mL for pravastatin and M1. The lower limit of quantitation, 0.500 ng/mL for all the analytes, was achieved when 50 microL of human serum was used. The intra- and inter-day precisions were within 7.4%, and the accuracy was between 95 and 103%. The on-line extraction was finished in 0.5 min and total analysis time was 2.5 min per sample.     

Quantitation of zopiclone and desmethylzopiclone in human plasma by high-performance liquid chromatography using fluorescence detection

A simple, reliable HPLC method using fluorescence detection (excitation 307 and emission 483 nm) was developed and validated for simultaneous quantitation of zopiclone and its metabolite desmethylzopiclone in human plasma. Following a single-step liquid-liquid extraction, the analytes and internal standard (zaleplon) were separated using an isocratic mobile phase on a reversed-phase C18 column. The lower limit of quantitation was 3 ng/mL for zopiclone and 6 ng/mL for desmethylzopiclone with a relative standard deviation of less than 5%. A linear dynamic range of 3-300 ng/mL for zopiclone and of 6-500 ng/mL for desmethylzopiclone was established. This HPLC method was validated with between-batch precision of 1.7-4.2% and 3.2-7.5% for zopiclone and desmethylzopiclone respectively. The between-batch accuracy was 99.4-111.5% and 101.6-104.8% for zopiclone and desmethylzopiclone, respectively. Frequently coadministered drugs did not interfere with the described methodology. Stability of zopiclone and desmethylzopiclone in plasma was excellent, with no evidence of degradation during sample processing (autosampler) and 30 days' storage in a freezer. This validated method is simple and repeatable enough to be used in pharmacokinetic studies.     

Enantiomeric determination of praziquantel and its main metabolite trans-4-hydroxypraziquantel in human plasma by cyclodextrin-modified micellar electrokinetic chromatography

A capillary electrophoresis method for the simultaneous quantitation of praziquantel and its main metabolite trans-4-hydroxypraziquantel enantiomers in human plasma was developed and validated using cyclodextrin-modified micellar electrokinetic chromatography. Sample clean-up involved a single-step liquid-liquid extraction of plasma with toluene after the addition of NaCl. The complete enantioselective analysis was obtained in less than 7 min using 2% w/v sulfated beta-cyclodextrin as chiral selector and 20 mmol/L sodium deoxycholate as surfactant, in 20 mmol/L sodium borate buffer, pH 10. A 50 microm x 42 cm uncoated fused-silica capillary was used for the analysis, performed at a voltage of 18 kV and at 20 degrees C. The calibration curves were linear over the 125-625 ng/mL concentration range. The mean recoveries for praziquantel and trans-4-hydroxypraziquantel were up to 96 and 71%, respectively, with good precision. All four enantiomers were quantified at two concentration levels (200 and 600 ng/mL) with precision and accuracy below 15%. The quantitation limit was 50 ng/mL for (-)-(R)- and (+)-(S)-praziquantel and 62.5 ng/mL for (-)-(R)- and (+)-(S)-trans-4-hydroxypraziquantel, using 1 mL of human plasma.     

Atmospheric pressure photoionization applied to quantitation of cyproterone acetate in human plasma

Cyproterone acetate [6-chloro-1beta,2beta-dihydro-17alpha-hydroxy- 3'H-cyclopropa(1,2)-pregna-1,4,6-triene-3,20-dione acetate] is a powerful antiandrogen used in the treatment of women suffering from disorders associated with androgenization such as hirsutism and acne. A fast, sensitive, and robustness method is developed for the determination and quantitation of cyproterone acetate in human blood plasma by liquid chromatography coupled with tandem mass spectrometry. Cyproterone acetate is extracted from 0.2 mL human plasma by liquid-liquid extraction. The method has a chromatographic run of 4.5 min, using a C18 analytical column (100- yen 2.1-mm i.d.), and the linear calibration curve over the range is linear from 1 to 500 ng/mL (r2 > 0.994). The between-run precision, based on the relative standard deviation replicate quality controls, is 96.2% (3 ng/mL), 97.5% (120 ng/mL), and 99.1% (400 ng/mL). The between-run accuracy was +/- 2.7%, 3.1%, and 4.8% for the previously mentioned concentrations, respectively. The method is employed in a bioequivalence study of two tablet formulations of cyproterone acetate (100 mg).     

A validated HPLC‐MS/MS method for determination of genipin‐1‐o‐glucuronic acid in rat plasma after administration of genipin and its application to a pharmacokinetic study

A sensitive and specific method was developed and validated for the quantitation of one major metabolite of genipin in rats plasma. The major metabolite was isolated from rat bile via semi‐preparative HPLC technology and its chemical structure was identified as genipin‐1‐o‐glucuronic acid (GNP‐GLU), which was for the first time used as a standard compound for quantitative analysis in rat plasma after administration of genipin. The application of high‐performance liquid chromatography–tandem mass spectrometry in negative mode in multiple reaction monitoring mode was investigated. Chromatographic separation was achieved on an Eclipse XDB‐C₁₈ column using a mobile phase consisting of water with 0.1% formic acid (A)–acetonitrile (B). The limit of detecation was 0.214 ng/mL and the lower limit of quantification was 0.706 ng/mL. The calibration curve was linear from 1.27 to 3810 ng/mL for plasma samples, with a correlation coefficient of 0.9924. The intra‐ and inter‐day precisions and accuracy were all within 15%. The recoveries of GNP‐GLU and puerarin were above 90.0 and 76.2%, respectively. The highly sensitive method was successfully applied to estimate pharmacokinetic parameters of GNP‐GLU following oral and intravenous administration of genipin to rats. Copyright © 2013 John Wiley & Sons, Ltd.     

Post-column reaction detection of biotin in human plasma ultrafiltrate based on laser-induced fluorescence energy transfer in the far-red spectral region

High performance liquid chromatography followed by post-column reaction detection in the far-red spectral region provides added sensitivity and selectivity. A homogeneous fluorescence energy transfer assay in the competitive mode based on the binding of biotin and streptavidin was developed as an on-line post-column reaction detection system. The labels used for energy transfer were R-Phycoerythrin conjugated to biotin and Cyanine 5 labeled with streptavidin. The energy transfer peak was measured at 670 nm and excitation was achieved using the 488 nm line of an argon ion laser. The biotin concentration in plasma ultrafiltrate ranged from 0.024 to 6.12 ng/mL (n = 6). The precision of the two controls, 0.24 and 2. 44 ng/mL, was found to be 18.70% and 9.92% relative standard deviation respectively. Accuracy was 10.47% and 1.95% difference from spiked, respectively (n = 6). The limit of detection was 21.70 pg/mL (8.90 x 10(-11)M) calculated based on a factor of 2x the standard deviation of the blank (n = 6). The correlation coefficient for the calibration curve was found to be 0.9995. Recovery from plasma ultrafiltrate at 2.44 ng/mL was 103.40% (n = 6). Detection selectivity was indicated by the absence of background fluorescence in six different plasma samples collected from six individual donors. Endogenous levels were detected in two of the six pools of plasma ultrafiltrates.     

Quantitation of 17alpha-ethinylestradiol in aquatic samples using liquid-liquid phase extraction, dansyl derivatization, and liquid chromatography/positive electrospray tandem mass spectrometry

A rapid and sensitive method for the analysis of 17alpha-ethinylestradiol (EE2) in environmental aqueous samples has been developed. Aquatic samples were extracted using liquid-liquid extraction, and organic phase extracts were concentrated and derivatized with dansyl chloride. Analysis was performed using high-performance liquid chromatography with positive electrospray ionization and tandem mass spectrometry (HPLC/ESI-MS/MS). Deuterated 17alpha-ethinylestradiol was used as internal standard and was added to samples before extraction. A limit of quantitation of 1 ng/L was obtained using a 25 mL aqueous sample. The average recovery of EE2 spiked into a 25 mL tapwater sample was 100%. This highly sensitive quantitation method is useful for measuring low levels of EE2 in aqueous environmental samples.     

Development of multi‐response optimization and quadratic calibration curve for determination of ten pesticides in complex sample matrices using QuEChERS dispersive liquid–liquid microextraction followed by gas chromatography

In this study, QuEChERS combined with dispersive liquid‐liquid microextraction is developed for extraction of ten pesticides in complex sample matrices of water and milk. In this regard, effective factors of proposed extraction technique combined with gas chromatography with flame ionization detector were designed, modeled, and optimized using central composite design, multiple linear regression, and Nelder–Mead simplex optimization. Later, univariate calibration model for ten pesticides was developed in concentration range of 0.5–100 ng/mL. Surprisingly, quadratic calibration behavior was observed for some of the pesticides. In this regard, Mandel's test was used for evaluating linearity and types of calibration equation. Finally, four pesticides followed linear calibration curve with sensitivity (0.23–0.66 mL/ng), analytical sensitivity (0.20–0.32), regression coefficient (0.988–0.995), limit of detection (0.39–1.83 ng/mL), and limit of quantitation (1.30–6.10 ng/mL) and six of them followed quadratic calibration curve with sensitivity (0.18–0.93 mL/ng), analytical sensitivity (0.25–0.86), regression coefficient (0.944–0.999), limit of detection (0.59–1.92 ng/mL), and limit of quantitation (1.96–6.40 ng/mL). The calculated limits of detection were below the maximum residue limits according to European Union pesticides database of European Commission. Finally, the proposed analytical method was used for determination of ten pesticides in water and milk samples.     

A novel approach for identification and measurement of hemoglobin adducts with 1,2,3,4-diepoxybutane by liquid chromatography/electrospray ionisation mass spectrometry and matrix-assisted laser desorption/ionisation tandem mass spectrometry

The structural characterisation of the adducts formed by in vitro interaction of hemoglobin (Hb) with 1,2,3,4-diepoxybutane (DEB), the most reactive 1,3-butadiene (BD) metabolite, was obtained by liquid chromatography/electrospray ionisation mass spectrometry (LC/ES-MS) analysis of modified tryptic peptides of human hemoglobin chains. The reactive sites of human hemoglobin towards DEB and its hydroxylated derivatives (trihydroxybutyl (THB)-derivatives) were identified through the characterisation of alkylated tryptic peptides by matrix-assisted laser desorption/ionisation tandem mass spectrometry (MALDI-MS/MS). Based on this characterisation, a procedure was set up to measure the Hb-adducts of THB-derivatives by isotope dilution mass spectrometry with the use of a deuterated peptide standard. The results obtained here could permit optimisation of molecular dosimetry of BD-adducts, and extension of the analysis to the biological monitoring of occupational exposure to butadiene.     

高效液相色谱-电喷雾离子阱串联质谱法检测芥子气经皮肤染毒SD大鼠肺中的DNA加合物

运用高效液相色谱-电喷雾离子阱串联质谱(HPLC-ESI-MS/MS)技术,建立了快速、简单、灵敏的SD大鼠肺中N7-(2-羟乙基硫代乙基)鸟嘌呤(N7-HETEG)的检测方法。以N7-苯甲基鸟嘌呤为内标,用甲醇和水为流动相进行梯度洗脱,正离子模式检测,方法的检出限(信噪比(S/N)≥10)为300 pg/mL,定量限(S/N≥20)为850 pg/mL。在300 pg/mL～1.28 μg/mL的质量浓度范围内,N7-HETEG浓度与N7-HETEG和内标的峰面积比呈良好的线性关系(线性相关系数为0.9929)。高、中、低3个添加水平的日内测定精密度(以相对标准偏差(RSD)计)和日间测定精密度均小于10%(n=7),回收率为100%～132%。对SD大鼠背部皮肤染芥子气,剂量分别为5.5、11、22和45 mg/kg,染毒4 d后检测大鼠肺脏中N7-HETEG的含量。各个不同染毒剂量下,每克组织中分别检测到（0.56±0.16）、（0.67±0.12）、（1.36±0.68）和(5.14±0.92) ng N7-HETEG, N7-HETEG的含量随着染毒剂量的增大而增大,表明N7-HETEG可用作芥子气暴露的体内生物标志物。