Rapid and sensitive determination of diacetylpolyamines in human fingernail by ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A rapid and sensitive ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) method has been developed and validated for quantitatively determining diacetylpolyamines in the human fingernail. N(1),N(8)-diacetylspermidine (DiAct-Spd), N(1),N(12)- diacetylspermine (DiAct-Spm) and 1,6-diaminohexane (DAH) the [internal standard (IS)] were extracted from human fingernail samples by MeOH: 5 M HCl solution, followed by 4-(N,N-dimethylaminosulfonyl)-7-fluoro- 2,1,3-benzoxadiazole (DBD-F) derivatization, and then separated on an ACQUITY BEH C18 column with a gradient elution of acetonitrile and water containing 0.1% formic acid. The derivatives of the diacetylpolyamines were fully separated within a short run time (3.0 min). The triple quadrupole mass spectrometric detection was performed in the multiple reactions monitoring (MRM) mode by the UPLC-ESI- MS/MS system in the positive ionization mode. MRM using the fragmentation transitions of m/z 455.20→ 100.07, 737.25 → 100.07 and 567.10 → 479.07 in the positive ESI mode was performed to quantify DiAct-Spd, DiAct-Spm and IS, respectively. The calibration curve is between 0.04 ng mL(-1) for DiAct-Spd and DiAct-Spm. The detection limits (signal to noise ratio of five) were 5-10 pg mL(-1). A good linearity was achieved from the calibration curves (r(2) >0.9999), and the intra-day and inter-day assay precisions were less than 7.06%. Furthermore, the recoveries (%) of the diacetylpolyamines spiked in the human fingernails were 79.18-97.11. The present method proved that the high sensitivity is characterized by the specificity and feasibility of the sample analysis. Consequently, the proposed method was used to analyze human fingernail samples from 15 lung- cancer patients and 22 healthy volunteers. Diacetylpolyamines were detected from the fingernails of the lung- cancer patients for the first time. The concentration of DiAct-Spd in the lung-cancer patient group tended to be higher than those in the healthy volunteers.

A quantitative analysis of the polyamine in lung cancer patient fingernails by LC-ESI-MS/MS.

A quantitative analysis of polyamines in lung cancer patient fingernails by the combination of 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole derivatives and liquid chromatography-electrospray ionization tandem mass spectrometry is described. The reaction of the reagent with eight kinds of polyamines, that is, N(1) -acetylputrescine (N(1) -actPUT), N(8) -acetylspermidine, N(1) -acetylspermine, 1,3-diaminopropane, putrescine (PUT), cadaverine, spermidine and spermine (SPM) effectively occurs at 60 °C for 30 min. The detection limits (signal-to-noise ratio 5) were 5-100 fmol. 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), that is, 1,6-diaminohexane, vs the injected amounts of polyamines (r(2) > 0.996), and the intra-day and inter-day assay precisions were <9.84%. Furthermore, the recoveries (%) of the polyamines spiked in the human fingernails were 89.14-110.64. The present method was applied to human fingernail samples from 17 lung cancer patients and 39 healthy volunteers. The polyamine concentration was different based on the gender, that is, the N(1) -actPUT and PUT contents were 3.10 times and 2.56 times higher in healthy men than in women, respectively. Additionally, in the lung cancer patient group, as compared with the healthy volunteers, the concentrations of SPM had a statistically significant (p < 0.05) correlation. Therefore, because the proposed method provides a good mass accuracy and the trace detection of the polyamines in human fingernails, this analytical technique could be a noninvasive technique to assist in the diagnosis and assessment of disease activity in lung cancer patients.

Rapid and sensitive determination of the intermediates of advanced glycation end products in the human nail by ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry.

The resolution of the intermediate advanced glycation end products (AGEs) in the human nail was carried out by the combination of 4,5-dimethyl-1,2-phenylenediamine (DMPD) derivatives and ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry (UPLC-ESI-TOF-MS). The reaction of the reagent with 3-deoxyglucosone (3-DG), methylglyoxal (MG), and glyoxal (GO) effectively proceeds at 60°C for 2h. The resulting derivatives were efficiently separated by a gradient program (a mixture of water and acetonitrile containing 0.1% formic acid) using a reversed-phase ACQUITY UPLC BEH C(18) column (1.7 µm, 50×2.1 mm i.d.) and sensitively detected by TOF-MS. The detection limits (signal-to-noise ratio=5) of the TOF-MS were 10 to 50 fmol. A good linearity was achieved from the calibration curve, which was obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS) (i.e., 2,3-hexanedione) versus the injected amounts of 3-DG, MG, and GO (r(2)>0.999), and the intra- and interday assay precisions were less than 6.89%. The derivatives of the compounds in the human nail were successfully identified by the proposed procedure. As we know, these three kinds of dicarbonyl intermediates in the formation of AGEs-3-DG, MG, and GO-were first found in human nail samples. Using these methods, the amounts of compound in the nails of healthy volunteers and diabetic patients were determined. When comparing the index from the diabetic patients with that from healthy volunteers, there is no significant difference in the content of the MG and GO in the nails. However, a statistically significant (P<0.001) correlation was observed between the 3-DG concentrations. Because the proposed method provides a good mass accuracy and the trace detection of the dicarbonyl intermediates of AGEs in the human nail, this analytical technique could be a noninvasive technique to assist in the diagnosis and assessment of disease activity in diabetic patients. Here we present a novel, sensitive, and simple method for the simultaneous determination of dicarbonyl compounds in the human nail.

First observation of N-acetyl leucine and N-acetyl isoleucine in diabetic patient hair and quantitative analysis by UPLC-ESI-MS/MS.

BACKGROUND: Type 2 diabetes patients (DP) have significantly higher plasma levels of valine, leucine, isoleucine and alanine than the controls. Specific amino acids may acutely and chronically regulate insulin secretion from the pancreatic ß-cells. We recently identified a metabolic signature of N-acetyl leucine (Ac-Leu) that strongly predicts diabetes development in mice hair. The Ac-Leu appears to be a potential biomarker candidate related to diabetes. However, the determination of Ac-Leu in human hair has not been reported. We measured the Ac-Leu, and its structure is similar to N-acetyl isoleucine (Ac-Ile) in human hair by ultra-performance liquid chromatography (UPLC) with electrospray ionization tandem mass spectrometry (ESI-MS/MS). The developed method was applied to the determination of Ac-Leu and Ac-Ile in the hair of healthy volunteers (HV) and DP. METHODS: Ac-Leu, Ac-Ile and N-acetyl norleucine (Ac-Nle, IS) were extracted from human hair samples by a micropulverized extraction procedure, then separated on a C18 column by isocratic elution of acetonitrile-0.1% formic acid in water:0.1% formic acid (14:86, vol./vol.). MRM using the fragmentation transitions of m/z 174.1→86.1 in the positive ESI mode was performed to quantify the N-acetyl leucine, N-acetyl isoleucine and IS. RESULTS: Ac-Leu, Ac-Ile and Ac-Nle in the human hair samples were completely separated by isocratic elution of a 5.0 min duration wash program using a reversed-phase column, and sensitively detected by LC-MS/MS in the ESI(+) MRM mode. The amounts of Ac-Leu and Ac-Ile in the hairs of HV and DP were determined. When comparing the concentrations between DP and those from HV, a statistically significant correlation was observed for the Ac-Leu (p<0.001) and Ac-Ile (p<0.01). CONCLUSIONS: The proposed method is useful for the determination of Ac-Leu and Ac-Ile in the hairs of DP and HV. Human hair may serve as a noninvasive biosample for the diagnosis of diabetes.

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.

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.