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The serum concentrations of creatinine (Cre) and urea are used for the determination of the renal function. However, the use of blood is not always suitable due to the invasive, hygienic and infection problems during its sample collection and handling. In contrast, saliva is relatively clean and the samples can be quickly and noninvasively collected and easily stored. Therefore, the simultaneous determination of Arginine (Arg), creatine (Cr) and Cre in the saliva of chronic kidney disease (CKD) patients was performed by UPLC-ESI-MS/MS together with the saliva of healthy volunteers. The evaluation of hemodialysis of CKD patients was also carried out by the determinations before and after the dialysis. An HS-F5 column was used for the simultaneous determination of Arg, Cr and Cre in the saliva. These molecules were rapidly separated within 4 min and sensitively determined by the multiple reaction monitoring (MRM) of the precursor ion [M+H](+) → product ions (m/z 175.1 → 70.1 for Arg; m/z 132.0 → 44.1 for Cr; m/z 114.0 → 44.1 for Cre). The concentration of Cre in the CKD patients was higher than that in the healthy persons. The concentrations of Cre in the saliva of the patients before hemodialysis were moderately correlated with the serum Cre concentrations (R(2) = 0.661). Furthermore, the concentration in the saliva obviously decreased after hemodialysis (before 0.73 mg/dL, after 0.25 mg/dL; p < 0.02). Thus, the proposed detection method using saliva by UPLC-MS/MS is useful for the evaluation of the renal function in CKD patients. The present method offers a new option for monitoring the hemodialysis of CKD patients., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Breast cancer is one of the most fearful diseases due to its increasing worldwide prevalence. A number of screening tests has been employed including clinical examinations and mammography. However, another screening method, which is a simple, not embarrassing, and low cost, is highly desired. Based on these findings, we are currently investigating the determination of polyamines including their acetylated structures for the diagnosis of breast cancer patients. We established a diagnostic approach to breast cancer patients based on the ratios of polyamines in saliva by a UPLC-MS/MS analysis., Methods: Twelve polyamines including their acetylated form were labeled with DBD-F, separated by a reversed-phase chromatography and detected by a Xevo TQ-S tandem mass spectrometer., Results: Eight polyamines (e.g., SPM, CAD, Ac-SPM, N1-Ac-SPD, N8-Ac-SPD) strongly correlated with the cancer patients. A simple 1-order equation was developed for the discrimination of the breast cancer patients and healthy persons (Y=0.5XSPM-3XAc-SPM-0.15XSPD-3.5XN8-Ac-SPD+0.5XN1-Ac-SPD+0.04XCAD). The concordance rate of the breast cancer patients and the healthy persons by the equation was 88% and 76% on the training set, respectively, whereas those on the validation set was both 88%. The score Y in the equation tended to correlate with the cancer stage of the patients and increased with the more serious conditions. The determination of polyamines in the saliva after the cancer patient operations was also performed to identify the concentration change before and after the surgical treatment. The discriminant analysis using 6 polyamines (i.e., N8-Ac-SPD, N1-Ac-SPD, CAD, DAc-SPD, PUT, and Ac-PUT), which were the most influenced molecules derived from the ROC analysis, was performed using the relative percentage. Both the sensitivity and specificity indicated nearly 80% from the ROC analysis result using the ratio of N8-Ac-SPD/(N1-Ac-SPD+N8-Ac-SPD)., Conclusion: The discrimination equation appears to be useful for the diagnosis of breast cancer patients. Furthermore, the ratio of N8-Ac-SPD/(N1-Ac-SPD+N8-Ac-SPD) may be adopted as an index of the health status after the surgical treatment., (Copyright © 2015 Elsevier B.V. All rights reserved.)

This paper proposes the dried saliva spot (DSS) as a convenient sampling technique for bioanalysis. The analytical method with the DSS was used for the determination of D,L-lactic acid (D,L-LA) and the D/L ratio of diabetic patients and prediabetic persons for the simple screening of the disease. The D,L-LA in the DSS was labeled with a chiral reagent (DMT-3(S)-Apy) for carboxylic acids and determined by UPLC-ESI-MS/MS. The limits of detection (signal-to-noise ratio (S/N) = 3) for the DSS analysis were on the amol level (∼30 amol). Because good stability, recovery, accuracy, and precision of the D,L-LA for the DSS method was also obtained from the proposed procedure, the DSS method was applied to the determination of the D- and L-isomers of LA of diabetic patients, and prediabetic and healthy persons. The D/L-LA ratio by the present DSS method and the HbA1c value in blood were well-correlated to the serious diabetic patients, whereas the relation in the prediabetic persons was not very good. The reason seems to be due to the rough saliva sampling, and not to the DSS method, because strict regulation was not requested for the prediabetic and healthy persons. In order to have a successful DSS analysis, the stability of the target molecule, the detection sensitivity to the target molecule, and the validated determination method are important.

A novel triazine-type chiral derivatization reagent, i.e., (S)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl) pyrrolidine-2-carboxylate (DMT-(S)-Pro-OSu), was developed for the highly sensitive and selective detection of chiral amines and amino acids by UPLC-MS/MS analysis. The enantiomers of amino acids were easily labeled with the reagents at room temperature within 40 min in an alkaline medium containing triethylamine. The diastereomers derived from proteolytic amino acids, except serine, were well separated under isocratic elution conditions by reversed-phase chromatography using an ODS column (Rs=1.2-9.0). DL-Serine was separated by use of an ADME column which has relatively higher polar surface than the conventional ODS column. The characteristic product ions, i.e., m/z 195.3 and m/z 209.3, were detected from all the diastereomers by the collision-induced dissociation of the protonated molecule. A highly sensitive detection on the amol-fmol level was obtained from the selected reaction monitoring (SRM) chromatogram. The chiral amines (e.g., adrenaline and noradrenaline) labeled with DMT-(S)-Pro-OSu were also well separated and sensitively detected by the present procedure. The method using DMT-(S)-Pro-OSu was used for the determination of DL-amino acids in the human saliva from healthy volunteers. Various L-amino acids were identified in the saliva. Furthermore, D-alanine (D-Ala) and D-proline (D-Pro) were also detected in relatively high concentrations (>5%). The ratio was higher in male saliva than in female saliva. However, the difference in the ratio of D-Ala for one day was not very high and the effect of foods and beverage seemed to be negligible. Based on the results using L-Ala-d3, the D-Ala in saliva seemed to be produced due to the racemization with some enzymes such as racemase. The racemization reaction was reversible, i.e., D-Ala-d3 was also racemized to L-Ala-d3 in saliva. Thus, care should be taken during the analysis of DL-amino acids in saliva. The present method using DMT-(S)-Pro-OSu may be applicable for the determination of chiral amine metabolomics, because the resulting derivatives produce the same product ions without relation to the compounds and show highly sensitive detection in the SRM mode of MS/MS. Consequently, DMT-(S)-Pro-OSu seems to be a useful chiral derivatization reagent for the determination of amines and amino acids in biological samples., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Novel triazine-type chiral derivatization reagents, i.e., (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine (DMT-3(S)-Apy) and (S)-4,6-dimethoxy-N-(pyrrolidin-3-yl)-1,3,5-triazin-2-amine (DMT-1(S)-Apy), were developed for the highly sensitive and selective detection of chiral carboxylic acids by UPLC-MS/MS analysis. Among the synthesized reagents, DMT-3(S)-Apy was a more efficient chiral reagent for the enantiomeric separation of chiral carboxylic acids in terms of separation efficiency by reversed-phase chromatography and detection sensitivity by ESI-MS/MS. The DMT-3(S)-Apy was used for the determination of 13 carboxylic acids in human saliva of healthy volunteers and diabetic patients. Various biological carboxylic acids including chiral carboxylic acids, and mono- and di-carboxylic acids were clearly identified in the saliva of healthy persons and diabetic patients. The concentrations of carboxylic acids detected in the saliva of diabetic patients were relatively higher than those in the healthy persons. Furthermore, the concentration of D-lactic acid (LA) and the ratio of D/L-LA in the diabetic patients were significantly higher than those in the healthy persons. The low ratio of D/L-LA in healthy persons was also identified to be independent of age and sex. These results suggest that the determination of the D/L-LA ratio in saliva might be applicable for the diagnosis of diabetes. Based on these observations, DMT-3(S)-Apy seems to be a useful chiral derivatization reagent for the determination not only of chiral carboxylic acids but also achiral ones. In conclusion, the proposed method using DMT-3(S)-Apy is useful for the carboxylic acid metabolomics study of various specimens.

Mass spectrometry has become a popular analytical tool because of its high sensitivity and specificity. The use of a chiral derivatization reagent for the mass spectrometry (MS) detection seems to be efficient for the enantiomeric separation of racemates. However, the number of chiral reagents for the liquid chromatography (LC)-MS/MS analysis is very limited. According to these observations, we are currently in the process of developing novel labeling reagents for chiral molecules in MS/MS analysis. The derivatization reagent that is effective for enhancing not only the electrospray ionization-MS/MS sensitivity but also the reversed-phase LC resolution of carboxylic acid enantiomers should have a highly proton-affinitive moiety and an asymmetric structure near the reactive functional group. Furthermore, the resulting derivative has to provide a characteristic product ion suitable for the selected reaction monitoring. Based upon these considerations, a series of prolylamidepyridines ((S)-N-pyrrolidine-2-carboxylic acid N-(pyridine-2-yl)amide (PCP2), (S)-N-pyrrolidine-2-carboxylic acid N-(pyridine-3-yl)amide, and (S)-N-pyrrolidine-2-carboxylic acid N-(pyridine-4-yl)amide) was synthesized as ideal labeling reagents for the enantioseparation of chiral carboxylic acids and evaluated in terms of separation efficiency and detection sensitivity by ultra-performance LC (UPLC)-MS/MS. Among the synthesized reagents, PCP2 was the most efficient chiral derivatization reagent for the enantioseparation of carboxylic acid. The Rs values and the detection limits of the derivatives of non-steroidal anti-inflammatory drugs, which were selected as the representative carboxylic acids, were in the range of 2.52-6.07 and 49-260 amol, respectively. The sensitive detection of biological carboxylic acids (detection limits, 32-520 amol) was also carried out by the proposed method using PCP2 and UPLC-MS/MS. The PCP2 was applied to the determination of carboxylic acids in human saliva. Several biological carboxylic acids, such as lactic acid (LA), 3-hydroxybutylic acid, maric acid, succinic acid, α-ketoglutalic acid, and citric acid, were clearly identified in the saliva of healthy persons and diabetic patients. Furthermore, the ratio of D-LA in diabetic patients was higher than that in normal subjects. Judging from these results, PCP2 seems to be a useful chiral derivatization reagent for the determination not only of chiral, but also achiral, carboxylic acids in real samples.

Background: In diabetes mellitus (DM) patients with ketoacidosis, ketone bodies, i.e., acetone, acetoacetic acid (AA) and β-hydroxybutyric acid (HA), are increased in the blood and urine. Acetone is also excreted by breathing due to the spontaneous decomposition of AA. Thus, the increase in acetone has been considered as one of the biomarkers for the diagnosis of DM. However, the determination of acetone in one's breath is not recommended because of the sample handling difficulty. We measured acetone in saliva by reversed-phase liquid chromatography (LC) with fluorescence (FL) detection. The proposed method was applied to the determination of acetone in the saliva of healthy volunteers and DM patients with and without ketoacidosis., Methods: 3-Pentanone (I.S.) and DBD-H in acetonitrile were added to freshly collected saliva and reacted at room temperature for 20 min in the presence of trifluoroacetic acid. After the reaction, the solution was centrifuged at 10,000 × g and 4 °C for 5 min. The supernatant was separated by reversed-phase LC and the FL detected at 550 nm (excitation at 460 nm)., Results: The concentrations of acetone in the DM patients with ketoacidosis were significantly higher than those of the normal subjects and DM patients without ketoacidosis. Furthermore, the total contents of the ketone bodies in the blood correlated with acetone in the saliva of the DM patients. The concentrations of acetone in the saliva of an emergency patient also correlated with the ketone bodies in the blood at each sampling time., Conclusion: The proposed method using LC-FL seems to be useful for the determination of acetone in the saliva of DM patients with ketoacidosis. The method offers a new option for the diagnosis and monitoring of DM patients with ketoacidosis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The determination of polyamines and their N-acetylated forms was performed by ultraperformance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). The polyamines efficiently reacted with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) in 0.1 M borax (pH 9.3) at 60 °C for 30 min. The resulting derivatives were analyzed by electrospray ionization (ESI)-MS and sensitively detected by selected reaction monitoring (SRM). Furthermore, a rapid separation of the polyamine derivatives within 10 min was performed by UPLC using an antipressurized column packed with 1.7-μm octadecylsilyl (ODS) silica gel. The limits of detection (S/N = 3) on the SRM chromatograms were at the attomole level (9-43 amol). This procedure was used to successfully determine 11 polyamines, including their N-acetylated forms, in the saliva of patients with primary and relapsed breast cancer and healthy volunteers. The level of several polyamines (Ac-PUT, Ac-SPD, Ac-SPM, DAc-SPD, and DAc-SPM) increases in breast cancer patients. Furthermore, the levels of three polyamines (Ac-SPM, DAc-SPD, and DAc-SPM) were significantly higher only in the relapsed patients. The present method proved highly sensitive and is characterized by specificity and feasibility for sample analysis. Consequently, the proposed method is useful for the noninvasive salivary diagnosis of cancer patients and could be applied to determine polyamines in several specimens of biological nature.

A simple and practical derivatization procedure for increasing the detectability and enantiomeric separation of chiral carboxylic acids in LC/ESI-MS/MS has been developed. (S)-Anabasine (ANA) was used as the derivatization reagent and rapidly reacted with carboxylic acids [3-hydroxypalmitic acid (3-OH-PA), 2-(β-carboxyethyl)-6-hydroxy-2,7,8-trimethylchroman (γ-CEHC), and etodolac] in the presence of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholium chloride. The resulting ANA-derivatives were highly responsive in ESI-MS operating in the positive-ion mode and gave characteristic product ions during MS/MS, which enabled sensitive detection using selected reaction monitoring; the detection responses of the ANA-derivatives were increased by 20-160-fold over those of the intact carboxylic acids and the limits of detection were in the low femtomole range (1.8-11 fmol on the column). The ANA-derivatization was also effective for the enatiomeric separation of the chiral carboxylic acids; the resolution was 1.92, 1.75, and 2.03 for 3-OH-PA, γ-CHEC, and etodolac, respectively. The derivatization procedure was successfully applied to a biological sample analysis; the derivatization followed by LC/ESI-MS/MS enabled the separation and detection of trace amounts of 3-OH-PA in neonatal dried blood spot and γ-CEHC in human saliva with a simple pretreatment and small sample volume., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A simultaneous determination method for the enantiomers of chiral carboxylic acids by the combination of ultraperformance liquid chromatography and mass spectrometry (UPLC-MS/MS) has been developed. (S)(+)-1-(2-Pyrrolidinylmethyl)-pyrrolidine (S-PMP) was used as the derivatization reagent for the high-throughput determination of biological chiral carboxylic acids, i.e., lactic acid (LA) and 3-hydroxybutyric acid (HA). The S-PMP efficiently reacted with the carboxylic acids under mild conditions at room temperature in the presence of 2,2'-dipyridyl disulfide and triphenylphosphine. The resulting S-PMP derivatives were highly responsive in the electrospray ionization (ESI)-MS operating in the positive-ion mode and gave characteristic product ions during the MS/MS, which enabled the sensitive detection using selected reaction monitoring. The derivatization was effective for the enantiomeric separation of the chiral carboxylic acids, and the resolution values of DL-LA and DL-HA were 4.91 and 9.37, respectively. Furthermore, a rapid separation of the derivatives of DL-LA and DL-HA within 7 min was performed using the UPLC system. The limits of detection on the column were in the low femtogram range (5-12 fg). The proposed procedure was successfully applied for the determination of the D- and L-isomers of LA and HA in the saliva of diabetes mellitus (DM) patients and healthy volunteers. The D-LA in DM patients was clearly higher than that in normal subjects. The derivatization followed by UPLC-ESI-MS/MS enabled the enantiomeric separation and detection of trace amounts of LA and HA in human saliva with a simple pretreatment and small sample volume.

The utility of Diels-Alder derivatization with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) for liquid chromatography/electrospray ionization tandem mass spectrometry of conjugated linoleic acids (CLAs) was examined. PTAD rapidly reacted with the CLAs, and the resulting derivatives were highly responsive in electrospray ionization mass spectrometry operating in the positive-ion mode. The derivatives produced characteristic product ions during tandem mass spectrometry, which enabled the sensitive detection [limit of detection 18 fmol (signal-to-noise ratio of 5)] and the identification of the conjugated diene position. The PTAD derivatization also significantly increased the reversed-phase liquid chromatography separation selectivity for the most biologically active CLA isomers: cis-9,trans-11-CLA and trans-10,cis-12-CLA. The PTAD derivatization was applied to analyses of food and biological samples; the major CLAs in milk and beef fat samples were successfully identified, and trace amounts of CLAs in human saliva were detected with a simple pretreatment and short analysis time.

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., (Copyright © 2011 John Wiley & Sons, Ltd.)

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., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Measurement of steroid levels in saliva has been proposed as a new laboratory tool for characterizing steroid metabolism, but it is not known whether the salivary levels of bile acids can be measured with accuracy and if so, whether such measurements provide information that is of clinical value. We developed and validated a sensitive and specific liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) method for the quantification of chenodeoxycholic acid (CDCA) and glycochenodeoxycholic acid (GCDCA), representative primary non-amidated and glycine-conjugated bile acids, in whole saliva. We also examined whether the salivary bile acid concentrations were dependent on the saliva flow rate, because this is a very important aspect in a discussion of the utility of salivary diagnostics. Saliva was deproteinized with ethanol and purified using a Strata-X cartridge. Bile acids were converted to their hydrazide derivatives using 2-hydrazinopyridine, and subjected to LC-MS/MS. Quantification was based on selected reaction monitoring using characteristic transitions, and deuterated CDCA and GCDCA were used as internal standards. This method allowed the reproducible and accurate quantification of the salivary bile acids using a 200-microl sample and the limits of quantification for CDCA and GCDCA were 25 and 50pg/ml, respectively. Using this method, the effect of increased saliva flow rate by gum-chewing on the salivary concentrations of CDCA and GCDCA was determined. The salivary level of GCDCA was significantly decreased by gum-chewing, whereas the concentration of CDCA remained constant. These results indicate that there is a good possibility that saliva may be a clinical tool for non-amidated bile acid testing.