Pentafluoropropionic Anhydride Derivatization and GC-MS Analysis of Histamine, Agmatine, Putrescine, and Spermidine: Effects of Solvents and Starting Column Temperature.

Gas chromatography-mass spectrometry (GC-MS) is useful for the quantitative determination of the polyamines spermidine (SPD) and putrescine (PUT) and of the biogenic amine agmatine (AGM) in biological samples after derivatization. This GC-MS method involves a two-step extraction with n-butanol and hydrochloric acid, derivatization with pentafluoropropionic anhydride (PFPA) in ethyl acetate, and extraction of the pentafluoropropionic (PFP) derivatives by toluene of SPD, PUT, and AGM. We wanted to extend this GC-MS method for the biogenic amine histamine (HA), but we faced serious problems that did not allow reliable quantitative analysis of HA. In the present work, we addressed this issue and investigated the derivatization of HA and the effects of toluene and ethyl acetate, two commonly used water-insoluble organic solvents in GC-MS, and oven temperature program. Derivatization of unlabelled HA (d0-HA) and deuterium-labelled HA (d4-HA) with PFPA in ethyl acetate (PFPA-EA, 1:4, v/v; 30 min, 65 °C) resulted in the formation of d0-HA-(PFP)2 and d4-HA-(PFP)2 derivatives. d4-HA and 13C4-SPD were used as internal standards for the amines after standardization. Considerable quantitative effects of toluene and ethyl acetate were observed. The starting GC column temperature was also found to influence considerably the GC-MS analysis of HA. Our study shows the simultaneous quantitative analysis of HA as HA-(PFP)2, AGM as AGM-(PFP)3, PUT as PUT-(PFP)2, and SPD as SPD-(PFP)3 derivatives requires the use of ethyl acetate for their extraction and injection into the GC-MS apparatus and a starting GC column temperature of 40 °C instead of 70 °C. The PFP derivatives of HA, AGM, PUT, and SPD were found to be stable in ethyl acetate for several hours at room temperature. Analytically satisfactory linearity, precision, and accuracy were observed for HA, AGM, PUT, and SPD in biologically relevant ranges (0 to 700 pmol). The limits of detection of AGM, PUT, and SPD were about two times lower in ethyl acetate compared to toluene (range, 1-22 fmol). The limits of detection were 1670 fmol for d0-HA and 557 fmol for d4-HA. Despite the improvements achieved in the study for HA, its analysis by GC-MS as a PFP derivative is challenging and less efficient than that of PUT, AGM, and SPD.

GC-MS Studies on the Conversion and Derivatization of γ-Glutamyl Peptides to Pyroglutamate (5-Oxo-Proline) Methyl Ester Pentafluoropropione Amide Derivatives.

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine, γ-Glu-Cys-Gly) is the most abundant intra-cellular dicarboxylic tripeptide with multiple physiological roles. In biological samples, glutathione exists in its reduced form GSH and in two stable oxidized forms, i.e., in its symmetric disulfide form GSSG and as S-glutathionyl residue in proteins. S-Glutathionylation is a post-translational modification, which is involved in several pathophysiological processes, including oxidative stress. The GSH-to-GSSG molar ratio is widely used as a measure of oxidative stress. γ-Glutamyl is the most characteristic structural moiety of GSH. We performed gas chromatography-mass spectrometry (GC-MS) studies for the development of a highly specific qualitative and quantitative method for γ-glutamyl peptides. We discovered intra-molecular conversion of GSH, GSSG, γ-Glu-Cys and of ophthalmic acid (OPH; γ-glutamyl-α-amino-n-butyryl-glycine) to pyroglutamate (pGlu; 5-oxo-proline, also known as pidolic acid) during their derivatization with 2 M HCl/CH3OH (60 min, 80 °C). For GC-MS analysis, the methyl esters (Me) were further derivatized with pentafluoropropionic (PFP) anhydride in ethyl acetate (1:4, v/v; 30 min, 65 °C) to their PFP derivatives. At longer reaction times, pGlu is hydrolyzed to Glu. Internal standards were prepared by derivatizing GSH, GSSG, γ-Glu-Cys and OPH in 2 M HCl/CD3OD. Quantification of the Me-PFP derivative of pGlu was performed in the electron-capture negative-ion chemical ionization (ECNICI) mode by selected-ion monitoring (SIM) of the mass-to-charge (m/z) ions 269 for unlabeled pGlu (d0Me-PFP-pGlu) and m/z 272 for the in situ prepared deuterium-labeled pGlu (d3Me-PFP-pGlu). Although not inherent to the analysis of small peptides, the present GC-MS method is useful to study several biochemical aspects of GSH. Using pentafluorobenzyl bromide (PFB-Br) as the derivatization reagent, we found that synthetic pGlu is converted in aqueous acetone (60 min, 50 °C) into its pentafluorobenzyl (PFB) ester (PFB-pGlu). This derivatization procedure is useful for the GC-MS analysis of free pGlu in the ECNICI mode. Quantitative analysis of PFB-pGlu by GC-MS requires the use of stable-isotope labeled analogs of pGlu as an internal standard.

Stable-Isotope Dilution GC-MS Measurement of Metformin in Human Serum and Urine after Derivatization with Pentafluoropropionic Anhydride and Its Application in Becker Muscular Dystrophy Patients Administered with Metformin, l-Citrulline, or Their Combination.

Metformin (N,N-dimethylguanylguanidine) is one of the most prescribed drugs with pleiotropic, exerted in part by not fully elucidated mechanisms of action. We developed and validated a gas chromatography-mass spectrometry (GC-MS) method for the quantitative analysis of metformin (metformin-d0) in 10-µL aliquots of human serum and urine using N,N-[dimethylo-2H6]guanylguanidine (metformin-d6) as the internal standard. The method involves evaporation of the samples to dryness, derivatization with pentafluoropropionic (PFP) anhydride in ethyl acetate (30 min, 65 °C), and extraction into toluene. The negative-ion chemical ionization GC-MS spectra of the PFP derivatives contain a single intense ion with mass-to-charge (m/z) ratios of m/z 383 for metformin-d0 and m/z 389 for metformin-d6. Our results suggest that all amine/imine groups of metformin-d0 and metformin-d6 are converted to their N,N,N-tripentafluoropropionyl derivatives, which cyclize to form a symmetric triazine derivative, of which the non-ring amine group is amidated. Quantification was performed by selected-ion monitoring (SIM) of m/z 383 and m/z 389. Upon validation, the method was applied to determine serum and urine metformin concentrations in 19 patients with Becker muscular dystrophy (BMD). Serum and urine samples were collected at baseline (Visit I), after six weeks of supplementation (Visit II) with metformin (3 × 500 mg/d; metformin group; n = 10) or l-citrulline (3 × 1500 mg/d; citrulline group; n = 9) followed by a six-week supplementation with 3 × 500 mg/d of metformin plus 3 × 1500 mg/d l-citrulline. At Visit I, the metformin concentration in the serum and urine was very low in both groups. The metformin concentrations in the serum and urine of the patients who first took metformin (MET group) were higher at Visit II and Visit III. The metformin concentration in the serum and urine samples of the patients who first took l-citrulline (CITR group) were higher at Visit III. The serum and urine concentrations of metformin were insignificantly lower in the CITR group at Visit III. The mean fractional excretion (FE) rate of metformin was 307% (Visit II) and 322% (Visit III) in the MET group, and 290% in the CITR group (Visit III). This observation suggests the accumulation of metformin in the kidney and its secretion in the urine. The GC-MS is suitable to measure reliably circulating and excretory metformin in clinical settings.

Two-Step Derivatization of Amino Acids for Stable-Isotope Dilution GC-MS Analysis: Long-Term Stability of Methyl Ester-Pentafluoropropionic Derivatives in Toluene Extracts.

Analysis of amino acids by gas chromatography-mass spectrometry (GC-MS) requires at least one derivatization step to enable solubility in GC-MS-compatible water-immiscible organic solvents such as toluene, to make them volatile to introduce into the gas chromatograph and thermally stable enough for separation in the GC column and introduction into the ion-source, and finally to increase their ionization by increasing their electronegativity using F-rich reagents. In this work we investigated the long-term stability of the methyl esters pentafluoropropionic (Me-PFP) derivatives of 21 urinary amino acids prepared by a two-step derivatization procedure and extraction by toluene. In situ prepared trideuteromethyl ester pentafluoropropionic derivatives were used as internal standards. GC-MS analysis (injection of 1 µL aliquots and quantification by selected-ion monitoring of specific mass fragments) was performed on days 1, 2, 8, and 15. Measured peak areas and calculated peak area ratios were used to evaluate the stability of the derivatives of endogenous amino acids and their internal standards, as well as the precision and the accuracy of the method. All analyses were performed under routine conditions. Me-PFP derivatives of endogenous amino acids and their stable-isotope labelled analogs were stable in toluene for 14 days. The peak area values of the derivatives of most amino acids and their internal standards were slightly higher on days 8 and 15 compared to days 1 and 2, yet the peak area ratio values of endogenous amino acids to their internal standards did not change. Our study indicates that Me-PFP derivatives of amino acids from human urine samples can easily be prepared, are stable at least for 14 days in the extraction solvent toluene, and allow for precise and accurate quantitative measurements by GC-MS using in situ prepared deuterium-labelled methyl ester as internal standard.

GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of N5-Carboxy-ornithine and N6-Carboxy-lysine.

Derivatization of amino acids by 2 M HCl/CH3OH (60 min, 80 °C) followed by derivatization of the intermediate methyl esters with pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C) is a useful two-step derivatization procedure (procedure A) for their quantitative measurement in biological samples by gas chromatography-mass spectrometry (GC-MS) as methyl ester pentafluoropropionic (PFP) derivatives, (Me)m-(PFP)n. This procedure allows in situ preparation of trideutero-methyl esters PFP derivatives, (d3Me)m-(PFP)n, from synthetic amino acids and 2 M HCl/CD3OD for use as internal standards. However, procedure A converts citrulline (Cit) to ornithine (Orn) and homocitrulline (hCit) to lysine (Lys) due to the instability of their carbamide groups under the acidic conditions of the esterification step. In the present study, we investigated whether reversing the order of the two-step derivatization may allow discrimination and simultaneous analysis of these amino acids. Pentafluoropropionylation (30 min, 65 °C) and subsequent methyl esterification (30 min, 80 °C), i.e., procedure B, of Cit resulted in the formation of six open and cyclic reaction products. The most abundant product is likely to be N5-Carboxy-Orn. The second most abundant product was confirmed to be Orn. The most abundant reaction product of hCit was confirmed to be Lys, with the minor reaction product likely being N6-Carboxy-Lys. Mechanisms are proposed for the formation of the reaction products of Cit and hCit via procedure B. It is assumed that at the first derivatization step, amino acids form (N,O)-PFP derivatives including mixed anhydrides. At the second derivatization step, the Cit-(PFP)4 and hCit-(PFP)4 are esterified on their C1-Carboxylic groups and on their activated Nureido groups. Procedure B also allows in situ preparation of (d3Me)m-(PFP)n from synthetic amino acids for use as internal standards. It is demonstrated that the derivatization procedure B enables discrimination between Cit and Orn, and between hCit and Lys. The utility of procedure B to measure simultaneously these amino acids in biological samples such as plasma and urine remains to be demonstrated. Further work is required to optimize the derivatization conditions of procedure B for biological amino acids.

GC-MS quantification of urinary symmetric dimethylarginine (SDMA), a whole-body symmetric l-arginine methylation index.

Circulating and excretory NG,N´G-dimethyl-l-arginine (symmetric dimethylarginine, SDMA) and NG,NG-dimethyl-l-arginine (asymmetric dimethylarginine, ADMA) are cardiovascular risk factors. Despite close chemical structures, the gas chromatography-mass spectrometry (GC-MS) measurement of SDMA is remarkably more difficult than that of ADMA for as yet unknown reasons. Here, we describe an improved GC-MS method for the quantitative determination of SDMA in human urine using commercially available NG,N´G-di-[2H3]methyl-l-arginine (d6-SDMA) as internal standard. The method is based on a single derivatization step with pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C) to N,N,N,O-tetrakis-pentafluoropropionyl derivatives, electron-capture negative-ion chemical ionization and selected-ion monitoring of the mass-to-charge (m/z) ions of m/z 456 for SDMA and m/z 462 for d6-SDMA.