Characterization of scallop midgut gland certified reference material for quantification of diarrhetic shellfish toxins.

A scallop midgut gland certified reference material, NMIJ CRM 7520-a, was developed for validation and quality assurance during the inspection of shellfish for diarrhetic shellfish toxins. The candidate material was prepared by using naturally-toxic and nontoxic boiled midgut glands spiked with okadaic acid (OA). The homogeneity and stability of the material were found to be appropriate. For the characterization of OA and dinophysistoxin-1 (DTX1), nine participants were involved in a co-laboratory study based on the Japanese Official Testing Method, where the compounds were assayed by liquid chromatography-tandem mass spectrometry following alkaline hydrolysis. The analytical values were obtained by the standard addition method with a standard spiking solution calibrated using the standard-solution certified reference materials OA and DTX1. The certified concentrations with expanded uncertainties (coverage factor k = 2, approximate 95% confidence interval) were determined to be (0.205 ±â€¯0.061) mg/kg for OA and (0.45 ±â€¯0.11) mg/kg for DTX1.

Interlaboratory comparison of liquid chromatography-tandem mass spectrometry quantification of diarrhetic shellfish toxins in scallop midgut glands.

An interlaboratory comparison (ILC) was organized as a measure of the analytical competency in the liquid chromatography-tandem mass spectrometry quantification of okadaic acid (OA) and dinophysistoxin-1 (DTX1) in scallop midgut gland samples. The test sample was prepared using boiled midgut glands of naturally contaminated scallops with DTX1 and its esters by spiking with OA, and homogeneity and stability of this test sample was assessed to be appropriate. Twenty laboratories participated in the ILC based on the Japanese official testing method; they submitted two sets of analytical concentrations of target analytes along with the details of their analytical protocols. For assessing these data, assigned values were established from another ILC where ten participants quantified the target analytes by the standard addition method. The mean analytical results of the former ILC showed good agreement with the assigned values, and the corresponding relative reproducibility standard deviations met the criterion of CODEX STAN 292. Meanwhile, the results of more than half of the participants were out of the uncertainty range of the assigned values; these participants were encouraged to investigate their protocols to improve their analytical capability.

A novel derivatization reagent possessing a bromoquinolinium structure for biological carboxylic acids in HPLC-ESI-MS/MS.

A novel bromoquinolinium reagent, i.e. 1-(3-aminopropyl)-3-bromoquinolinium bromide (APBQ), was synthesized for the analysis of carboxylic acids. A simple and practical precolumn derivatization procedure using the APBQ in RP chromatography and MS (HPLC-MS) has been developed using bile acids and free fatty acids, as the representative carboxylic acids in biological samples. The APBQ efficiently reacted with carboxylic acids at 60°C for 60 min in the presence of N,N-dicyclohexylcarbodiimide and pyridine as the activation reagents. Because the APBQ possesses a bromine atom in the structure, the identification of a series of carboxylic acids was easily achieved due to the characteristic bromine isotope pattern in the mass spectra. The APBQ also has a quaternary amine structure, thus the positively charged derivatives are predominate for the highly sensitive detection of carboxylic acids. The APBQ was successfully applied to the selective determination of biological carboxylic acids in human plasma. The bile acids (chenodeoxycholic acid and deoxycholic acid) and several saturated (stearic acid and palmitic acid) and unsaturated free fatty acids (oleic acid and linoleic acid) were reasonably determined by HPLC-MS under the proposed procedure. Based on the results of analyses of human plasma and saliva, the proposed procedure using APBQ seems to be applicable for the qualitative and quantitative analyses of a series of carboxylic acids in biological samples.

Characterization of certified reference material for the quantification of water in bioethanol.

The National Metrology Institute of Japan has issued a certified reference material of bioethanol (NMIJ CRM 8301-a) for the quantification of water, methanol, sulfur, and copper. This paper presents technical details for the characterization of the water in NMIJ CRM 8301-a. The characterization was performed using coulometric and volumetric Karl-Fischer (KF) titrations. To reduce moisture absorption, sample handling and KF titration were performed in a glove box under a dried nitrogen atmosphere. In addition, a rubber cap with a three-way valve was attached to the ampoule immediately after opening so as to minimize the influence of moisture. Sample aliquots were obtained using a gas-tight microsyringe through the valve, and injected into the KF cell as soon as possible. The certified value of water obtained from coulometric and volumetric KF titrations was 1.688 mg g(-1), and the expanded uncertainty (coverage factor, k = 2) was 0.028 mg g(-1). This CRM would be suitable for the monitoring of water in bioethanol and similar matrices.

Rapid enantiomeric separation and simultaneous determination of phenethylamines by ultra high performance liquid chromatography with fluorescence and mass spectrometric detection: application to the analysis of illicit drugs distributed in the Japanese market and biological samples.

A rapid enantiomeric separation and simultaneous determination method based on ultra high performance liquid chromatography (UHPLC) was developed for phenethylamine-type abused drugs using (R)-(-)-4-(N,N-dimethylaminosulfonyl)-7-(3-isothiocyanatopyrrolidin-1-yl)-2,1,3-benzoxadiazole ((R)-(-)-DBD-Py-NCS) as the chiral fluorescent derivatization reagent. The derivatives were rapidly enantiomerically separated by reversed-phase UHPLC using a column of 2.3-µm octadecylsilica (ODS) particles by isocratic elution with water-methanol or water-acetonitrile systems as the mobile phase. The proposed method was applied to the analysis of products containing illicit drugs distributed in the Japanese market. Among the products, 1-(3,4-methylenedioxyphenyl)butan-2-amine (BDB) and 1-(2-methoxy4,5-methylenedioxyphenyl)propan-2-amine (MMDA-2) were detected in racemic form. Furthermore, the method was successfully applied to the analysis of hair specimens from rats that were continuously dosed with diphenyl(pyrrolidin-2-yl)methanol (D2PM). Using UHPLC-fluorescence (FL) detection, (R)- and (S)-D2PM from hair specimens were enantiomerically separated and detected with high sensitivity. The detection limits of (R)- and (S)-D2PM were 0.12 and 0.21 ng/mg hair, respectively (signal-to-noise ratio (S/N) = 3).

Amino acid analysis via LC-MS method after derivatization with quaternary phosphonium.

(5-N-Succinimidoxy-5-oxopentyl)triphenylphosphonium bromide (SPTPP) is a highly sensitive and positively charged precolumn derivatization reagent for the analysis of amino acids in liquid chromatography-electrospray ionization-tandem mass spectrometry. The synthesis of this reagent and handling of the derivatization reaction are quite simple. It reacts with amino acids rapidly and with high efficiency. MS/MS analysis revealed that the SPTPP-derivatized amino acids formed strong product ions; thus, highly sensitive and selective detection is possible in the selected reaction monitoring mode. The limits of detection for the SPTPP-derivatized amino acids are in the sub-fmol range. The sensitivities of the derivatized amino acids increased about 500-fold, as compared to those of underivatized amino acids.

Determination of DL-amino acids, derivatized with R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole, in nail of diabetic patients by UPLC-ESI-TOF-MS.

The resolution of free DL-amino acids in human nail was carried out by combination of the R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS] derivatives and UPLC-ESI-TOF-MS. The reaction of the reagent with amino acids effectively proceeds at 55 °C for 20 min in the presence of 1% triethylamine (TEA) to produce the corresponding diastereomers. Each pair of the resulting derivatives was efficiently separated by a gradient program (a mixture of H(2)O and CH(3)CN containing 0.1% formic acid (HCOOH) or 5 mM CH(3)COONH(4) and CH(3)CN) using a reversed-phase ACQUITY UPLC™ BEH C(18) (1.7 µm, 100 mm × 2.1 mm i.d.) column and sensitively detected by TOF-MS. The detection limits (S/N=3) of the TOF-MS were 1.0-750 fmol, respectively. A good linearity was achieved from the calibration curves, which was obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), i.e., 6-aminohexanoic acid, versus the injected amounts of each amino acid (r(2)>0.996), and the intra-day and inter-day assay precisions were less than 8.93%. The derivatives of the free DL-amino acids in human nail were successfully identified by the proposed procedure. As we know, for the first time, these five kinds of D-amino acids, which were D-Ala, D-Val, D-Pro, D-Ile and D-Leu, were found from human nail samples. Fifteen kinds of L-amino acids were also recognized from human nails. Using these methods, the amounts of DL-amino acids in the nails of healthy volunteers and diabetic patients were determined. When comparing the index from diabetic patients to those from healthy volunteers, there is no significant difference in the content of the L-amino acids in the nails. However, a statistically significant (P<0.01) correlation was observed between the D/L-amino acid concentration ratios (Ala, Val, Ile, Leu). Therefore, because the proposed method provides a good mass accuracy and the trace detection of the DL-amino acids in human nails, this analytical technique could be a noninvasive technique to assist in the diagnosis and assessment of disease activity in diabetic patients.

Stable isotope-dilution liquid chromatography/tandem mass spectrometry method for determination of thyroxine in saliva.

A liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method for the determination of thyroxine (T(4)) in human saliva has been developed and validated. The saliva was deproteinized with methanol, purified using a Strata-X™ cartridge, and subjected to LC/ESI-MS/MS. Quantification was based on selected reaction monitoring, and [(13)C(6)]-T(4) was used as the internal standard. This method allowed the reproducible (intra- and inter-assay relative standard deviations, <4.8%) and accurate (analytical recovery, 96.5-99.6%) quantification of the salivary T(4) using a 400 µl sample, and the limit of quantification was 25.0 pg/ml. A preliminary study using the developed method found that there is a diagnosable difference in the salivary T(4) concentration between the euthyroid subjects and the patients with Graves disease.

Development and validation of stable-isotope dilution liquid chromatography-tandem mass spectrometric method for determination of salivary progesterone.

A method for the quantification of progesterone (PROG) in human saliva using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata™-X cartridge, and subjected to LC-ESI-MS/MS. Quantification was based on selected reaction monitoring, and deuterated PROG was used as the internal standard. This method allowed the reproducible (intra- and inter-assay relative standard deviations, <2.2%) and accurate (analytical recovery, 96.6-99.7%) quantification of the salivary PROG using a 400 µL sample, and the limit of quantification was 12.5 pg/mL. The developed method enabled detection of the variation in the salivary PROG concentrations of healthy volunteers during the menstrual cycle and measurement of the salivary concentrations of pregnant women. The method is expected to be an alternative to the blood PROG monitoring in clinical examinations, because saliva collection is easy, non-invasive and repeatable.

A specific LC/ESI-MS/MS method for determination of 25-hydroxyvitamin D3 in neonatal dried blood spots containing a potential interfering metabolite, 3-epi-25-hydroxyvitamin D3.

Vitamin D deficiency in an infant is associated with a wide range of adverse health outcomes in later life. A method for the quantification of 25-hydroxyvitamin D(3) [25(OH)D(3), the best-established indicator of vitamin D status] in neonatal dried blood spots (DBSs) using LC/ESI-MS/MS has been developed and validated. The method employed two steps of derivatization, a Diels-Alder reaction with 4-phenyl-1,2,4-triazoline-3,5-dione followed by acetylation, to enhance the detectability of 25(OH)D(3) in ESI-MS/MS and to separate 25(OH)D(3) from 3-epi-25-hydroxyvitamin D(3) [3-epi-25(OH)D(3)], a potent interfering metabolite. 25(OH)D(3) was extracted from two DBS punches (3 mm in diameter, equivalent to 5.3 µL of whole blood), purified using an Oasis HLB(®) cartridge, and subjected to derivatization prior to analysis with LC/ESI-MS/MS. 25-Hydroxyvitamin D(4) was used as the internal standard. This method was reproducible (intra- and inter-assay RSDs, <6.9%) and accurate (analytical recovery, 95.2-102.7%), and the LOQ was 3.0 ng/mL. The developed method enabled specific quantification of 25(OH)D(3) in neonatal DBSs and detection of vitamin D deficiency without interference from 3-epi-25(OH)D(3).

Simultaneous determination of polyamines in human nail as 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole derivatives by nano-flow chip LC coupled with quadrupole time-of-flight tandem mass spectrometry.

BACKGROUND: Polyamines are active biogenic amines which play an important role in cell growth and proliferation and the synthesis of proteins and nucleosides. In recently studies, rapid tumor growth has been associated with markedly altered polyamine biosynthesis and accumulation. Therefore, the accurate detection and identification of all the polyamines simultaneously in a single analysis is becoming more and more important for the study of their biochemical roles. METHODS: The development of a simultaneous determination method for 9 polyamines in human nails was attempted by the combination of nano-flow chip LC and quadrupole time-of-flight tandem mass spectrometry (Q-TOF-MS/MS). The proposed method was for the determination in the nails of healthy persons and cancer patients. RESULTS: The derivatives of the polyamines in human nail were completely separated by a gradient of an 18 min duration wash program using a reversed-phase chip column. The polyamine derivatives were then introduced into the Q-TOF-MS/MS instrument and sensitively detected in the ESI(+) mode. Polyamine concentration was different based on the gender, i.e., PUT and SPD is higher in men than in women in the healthy persons. Additionally, in the lung cancer patients group, as compared with the healthy persons, concentrations of PUT, N¹-actPUT, and SPM were significantly increased. CONCLUSIONS: We here present a novel sensitive, simple method for the simultaneous determination of polyamines in human nails. Furthermore, we show that polyamines (ORN, DAP, CAD, N¹-actPUT, N8-actSPD, N¹-actSPM) were detected from the human nails. Human nails may may serve as the noninvasive bio sample for diagnosis of the chronic disease.

Biomarker discovery in biological specimens (plasma, hair, liver and kidney) of diabetic mice based upon metabolite profiling using ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry.

BACKGROUND: The number of diabetic patients has recently been increasing worldwide. Diabetes is a multifactorial disorder based on environmental factors and genetic background. In many cases, diabetes is asymptomatic for a long period and the patient is not aware of the disease. Therefore, the potential biomarker(s), leading to the early detection and/or prevention of diabetes mellitus, are strongly required. However, the diagnosis of the prediabetic state in humans is a very difficult issue, because the lifestyle is variable in each person. Although the development of a diagnosis method in humans is the goal of our research, the extraction and structural identification of biomarker candidates in several biological specimens (i.e., plasma, hair, liver and kidney) of ddY strain mice, which undergo naturally occurring diabetes along with aging, were carried out based upon a metabolite profiling study. METHODS: The low-molecular-mass compounds including metabolites in the biological specimens of diabetic mice (ddY-H) and normal mice (ddY-L) were globally separated by ultra-performance liquid chromatography (UPLC) using different reversed-phase columns (i.e., T3-C18 and HS-F5) and detected by electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). The biomarker candidates related to diabetes mellitus were extracted from a multivariate statistical analysis, such as an orthogonal partial least-squares-discriminant analysis (OPLS-DA), followed by a database search, such as ChemSpider, KEGG and HMDB. RESULTS: Many metabolites and unknown compounds in each biological specimen were detected as the biomarker candidates related to diabetic mellitus. Among them, the elucidation of the chemical structures of several possible metabolites, including more than two biological specimens, was carried out along with the comparison of the tandem MS/MS analyses using authentic compounds. One metabolite was clearly identified as N-acetyl-L-leucine based upon the MS/MS spectra and the retention time on the chromatograms. CONCLUSIONS: N-acetyl-L-leucine is an endogenous compound included in all biological specimens (plasma, hair, liver and kidney). Therefore, this metabolite appears to be a potential biomarker candidate related to diabetes. Although the structures of other biomarker candidates have still not yet determined, the present approach based upon a metabolite profiling study using UPLC-ESI-TOF-MS could be helpful for understanding the abnormal state of various diseases.

Simultaneous and group determination methods for designated substances by HPLC with multi-channel electrochemical detection and their application to real samples.

Many psychotropic substances are illegally available on the streets and/or via the Internet. This wide distribution has become a serious social problem. To control this problem, many substances have been controlled as 'designated substances' (Shitei-Yakubutsu) in Japan since April 2007 by the Pharmaceutical Affairs Law, including tryptamines, phenethylamines and piperazines. In the present study, simultaneous determination methods using HPLC with multi-channel electrochemical detection (MECD) were developed for the designated substances. The proposed methods utilizing online electrochemical oxidation are the first report on the simultaneous determination of various designated substances. The methods involve direct determination and require no complicated pretreatments such as fluorescence labeling. The designated substances were separated by reversed-phase chromatography using a TSK-gel ODS-100V (4.6 × 250 mm, i.d., 3 µm) and gradient elution by a mixture of potassium phosphate buffer, methanol and acetonitrile. The total separation of 31 designated substances was successfully performed but required long chromatographic run times. Thus, the designated substances were divided into three groups: (1) tryptamines, (2) phenethylamines and (3) piperazines and others. They were then analyzed by HPLC-MECD as another separation method. The suitable applied voltages for each designated substance were determined based upon the hydrodynamic voltammogram. The limits of detection (signal-to-noise ratio of 3) of the designated substances for the most suitable voltages were in the range of 17.1 pg (5-MeO-MIPT) to 117 ng (indan-2-amine). The calibration curves based on the peak heights were linearly related to the amounts of the designated substances (R(2) > 0.999). Good accuracy and precision by intra-day assay and inter-day assay were also obtained using the present procedures. The proposed methods were applied to the analyses of the designated substance in several real samples.

HPLC enantioseparation of α,α-diphenyl-2-pyrrolidinemethanol and methylphenidate using a chiral fluorescent derivatization reagent and its application to the analysis of rat plasma.

Enantioseparation of α,α-diphenyl-2-pyrrolidinemethanol (D2PM) and methylphenidate (MPH; Ritalin(®)) using (R)-(-)-4-(N,N-dimethylaminosulfonyl)-7-(3-isothiocyanatopyrrolidin-1-yl)-2,1,3-benzoxadiazole as the chiral derivatization reagent has been achieved for the first time, and a simple, reliable detection method using HPLC with fluorescence detection has been developed. D2PM and MPH have been derivatized with (R)-(-)-4-(N,N-dimethylaminosulfonyl)-7-(3-isothiocyanatopyrrolidin-1-yl)-2,1,3-benzoxadiazole at 55°C for 15 min. The derivatives of D2PM and MPH have been separated, completely and rapidly, using a reversed-phase system within 16 min (resolution factor (R(s))=1.60 and 2.53, respectively). The detection limits of (R)- and (S)-D2PM were found to be 6.8 and 13 ng/mL, respectively, and those of D- and L-threo-MPH were 61 and 66 ng/mL, respectively (S/N=3). The proposed method was successfully applied to the analysis of rat plasma, where the rats were separately dosed with D2PM and MPH (Ritalin).

Practical analytical approach for the identification of biomarker candidates in prediabetic state based upon metabonomic study by ultraperformance liquid chromatography coupled to electrospray ionization time-of-flight mass spectrometry.

The number of diabetic patients has recently been increasing worldwide. Thus, the discovery of potential diabetic biomarker(s), leading to the early detection and/or prevention of diabetes mellitus, is strongly required. The diagnosis of the prediabetic state in humans is a very difficult issue because of the lifestyle differences in each person and ethical consideration. Upon the basis of these considerations, animal experiments using ddY strain mice (ddY-H), which undergo naturally occurring diabetes along with age, were carried out in this study. Biomarker discovery based upon a metabonome study is now quite common, the same as that in the proteome analysis. Reversed-phase liquid chromatography-mass spectrometry (LC-MS) has mainly been used for the extensive analysis of low-molecular mass compounds including metabolites. The metabolites in the plasma of diabetic mice (ddY-H) and normal mice (ddY-L) were exhaustively separated and detected by ultraperformance liquid chromatography along with electrospray ionization time-of-flight mass spectrometry (UPLC-ESI-TOF-MS) using T3-C18 and HS-F5 columns. The biomarker candidates related to diabetes mellitus were extracted from the metabolite profiling of ddY-H and ddY-L at 5, 9 13, and 20 weeks old using a multivariate statistical analysis such as orthogonal partial least-squares-discriminant analysis (OPLS-DA). Various metabolites and unknown compounds were detected as biomarker candidates related to diabetic mellitus. Furthermore, the concentration of several metabolites on Lysine biosynthesis and Lysine degradation pathways were remarkably changed between the 9-week old ddY-H and ddY-L mice. Because a couple of biomarker candidates related to the prediabetic state were identified using the present approach, the metabolite profiling study could be helpful for understanding the abnormal state of various diseases.

Highly sensitive and positively charged precolumn derivatization reagent for amines and amino acids in liquid chromatography/electrospray ionization tandem mass spectrometry.

We have developed a highly sensitive and positively charged precolumn derivatization reagent, (5-N-succinimidoxy-5-oxopentyl)triphenylphosphonium bromide (SPTPP), for amines and amino acids in liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). The handling of the derivatization reaction is quite simple and the reagent reacts with the analytes rapidly and with high efficiency. The derivatized analytes were observed to form regular and intense product ions upon MS/MS analysis; thus, highly sensitive and selective detection was possible in the selected reaction monitoring (SRM) mode. The limits of detection of the SPTPP-derivatized analytes were less than sub-femtomole levels. The sensitivities of the derivatized analytes increased about 500-fold compared to those of underivatized analytes. Since the hydrophobicities of the samples increased after their derivatization, the resolution of the analytes improved dramatically when a reversed-phase system was used. The relative standard deviations of intra-day and inter-day variations were below 10.6% and 13.3%, respectively. The accuracy ranged between 86.6-113% and 83.4-113%, respectively. Furthermore, the developed reagent was used for the analysis of the neurotransmitter 4-aminobutanoic acid (GABA) and oxidative stress markers such as oxidized, nitrated, and halogenated tyrosines in rat serum.

Simple and practical derivatization procedure for enhanced detection of carboxylic acids in liquid chromatography-electrospray ionization-tandem mass spectrometry.

A simple and practical derivatization procedure for increasing the detection responses of carboxylic acids in liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) has been developed. 2-Hydrazinopyridine (HP) and 2-picolylamine (PA) rapidly reacted with biologically and clinically important carboxylic acids [chenodeoxycholic acid, glycochenodeoxycholic acid, prostaglandin E2, 2-(-carboxyethyl)-6-hydroxy-2,7,8-trimethylchroman (gamma-CEHC),alpha-lipoic acid, homovanillic acid (HVA) and 5-hydroxyindole-3-acetic acid] in the presence of 2,2'-dipyridyl disulfide and triphenylphosphine. The resulting HP- and PA-derivatives were highly responsive in ESI-MS operating in the positive-ion mode and gave characteristic product ions during MS/MS, which enabled the sensitive detection using selected reaction monitoring. Among the two reagents, PA was of more practical use; the detection responses of the PA-derivatives were increased by 9-158-fold over the intact carboxylic acids and the limits of detection were in the low femtomole range (1.5-5.6 fmol on column). The PA-derivatization was successfully applied to a biological sample analysis; the derivatization followed by LC-ESI-MS/MS enabled the detection of trace amounts of bile acids, gamma-CEHC and HVA in human saliva with a simple pretreatment, small sample volume and short analysis time.

Rapid, sensitive and simultaneous determination of fluorescence-labeled designated substances controlled by the Pharmaceutical Affairs Law in Japan by ultra-performance liquid chromatography coupled with electrospray-ionization time-of-flight mass spectrometry.

A simultaneous determination method based on ultra-performance liquid chromatography (UPLC) with fluorescence (FL) detection and electrospray-ionization time-of-flight mass spectrometry (ESI-TOF-MS) was developed for 16 "designated substances" (Shitei-Yakubutsu) controlled by the Pharmaceutical Affairs Law in Japan. These substances were first labeled with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole at 60 degrees C for 2 h in 0.1 M borax (pH 9.3). The resulting fluorophores were well separated by reversed-phase chromatography using an Acquity UPLC BEH C(18) column (1.7 microm, 100 mm x 2.1 mm i.d.) by isocratic elution with a mixture of water and acetonitrile-methanol (20:80) containing 0.1% formic acid. The separated derivatives were sensitively detected by both FL and TOF-MS. However, the determination of several designated substances by FL detection showed interference from endogenous substances in biological samples. Therefore, the determination in real samples was carried out by a combination of UPLC separation and ESI-TOF-MS detection. The structures of the designated substances were identified from the protonated-molecular ions [M+H](+) obtained from the TOF-MS measurement. The calibration curves obtained from the peak area ratios of the internal standard (I.S.), i.e., 3-phenyl-1-propylamine, and the designated substances versus the injection amounts showed good linearity. The limits of detection (S/N = 3) and the limits of quantification (S/N = 10) in 0.1 mL of human plasma and urine for the present method were 0.30-150 pmol and 1.0-500 pmol, respectively. Good accuracy and precision (according to intraday and interday assays) were also obtained with the present procedure. This method was applied to analyses of human plasma, urine and real products.

Rapid determination of oxidized methionine residues in recombinant human basic fibroblast growth factor by ultra-performance liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry with in-source collision-induced dissociation.

The primary structure of the deteriorated recombinant human basic fibroblast growth factor (rhbFGF) was determined by ultra-performance liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC/ESI-QTOF-MS) with in-source collision-induced dissociation (CID). The rhbFGFs before and after treatment with hydrogen peroxide (H(2)O(2)) were separated using an ACQUITY UPLC BEH300 C18 column (1.7 microm, 150 mm x 2.1 mm i.d.) with a gradient elution of a mixture of water/acetonitrile containing 0.1% formic acid. The separated proteins were then detected by a SYNAPT High Definition Mass Spectrometry system (SYNAPT-MS). Two methionine (Met) residues in the rhbFGF structure were oxidized to Met-sulfoxide (Met-O) in 0.03% H(2)O(2) at pH 2.0. As the result, three peaks, except for the peak of rhbFGF, appeared on the chromatogram. The three proteins corresponding to each peak were estimated as the denatured rhbFGFs including the Met-O residue(s) with TOF-MS. Furthermore, the position of the Met-O residue(s) was efficiently identified by UPLC/ESI-QTOF-MS using the in-source CID technique. The proposed method seems to be very useful for the structural elucidation of proteins, because the oxidized Met residues in rhbFGF were easily and rapidly identified.

Rapid detection of ketamine and norketamine in rat hair using micropulverized extraction and ultra-performance liquid chromatography-electrospray ionization mass spectrometry.

A new method for the rapid and simultaneous detection of ketamine and its major metabolite, norketamine, in rat hair has been developed by combining micropulverized extraction and ultraperformance liquid chromatography-electrospray ionization mass spectrometry. By using reversed-phase UPLC, ketamine and norketamine were well separated within 2 min. Using ketamine-dosed rat hair, the conditions for micropulverized extraction were optimized, and the limits of detection and quantification of the developed method were found to be 1.7 and 5.7 pg/mg hair for ketamine, respectively. The precisions achieved with this method were slightly better than that obtained with conventional acidic methanol extraction method. Using this proposed method, analysis of the washed rat hair could be completed within 16-17 min. This method is expected to be applied for the analysis of the hair samples of not only rats but also ketamine abusers.