Pentafluoropropionic Anhydride Research Articles

Applications of Sample Preparation Techniques in the Analysis of New Psychoactive Substances

The global rise of new psychoactive substances (NPSs) poses challenges for their analysis in biological matrices due to their complex chemistries and short market lifespan. A comparative study for the simultaneous extraction, separation, and detection of 19 NPSs was conducted. Six solid-phase extraction (SPE) methods and one supported liquid extraction method (SLE) were compared for the extraction of analytes from blood, serum, plasma, and urine. Comparisons of four derivatization agents were conducted, at four temperatures and two incubation times. Extraction methods were assessed by precision, sensitivity, and extraction efficiency. Derivatizing agents were assessed on their selectivity and sensitivity, and a three-way ANOVA was conducted to determine statistical significance. CSDAU SPE cartridges were shown to be the most efficient when extracting analytes from blood, serum, and plasma, whereas Xcel I cartridges performed the strongest when extracting analytes from urine. SPE extraction efficiencies, when utilizing the best-performing cartridges, ranged from 49 to 119%. SLE successfully extracted all analytes from all matrices (ranging from 22 to 120%). Pentafluoropropionic anhydride: ethyl acetate was the most successful derivatizing agent, allowing all analytes to be detected, with the highest peak area responses and more unique spectra. The optimum temperature for incubation was 37 °C, with no statistical difference found between the two incubation times.

Quality Control in Targeted GC-MS for Amino Acid-OMICS.

Gas chromatography-mass spectrometry (GC-MS) is suitable for the analysis of non-polar analytes. Free amino acids (AA) are polar, zwitterionic, non-volatile and thermally labile analytes. Chemical derivatization of AA is indispensable for their measurement by GC-MS. Specific conversion of AA to their unlabeled methyl esters (d0Me) using 2 M HCl in methanol (CH3OH) is a suitable derivatization procedure (60 min, 80 °C). Performance of this reaction in 2 M HCl in tetradeutero-methanol (CD3OD) generates deuterated methyl esters (d3Me) of AA, which can be used as internal standards in GC-MS. d0Me-AA and d3Me-AA require subsequent conversion to their pentafluoropropionyl (PFP) derivatives for GC-MS analysis using pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C). d0Me-AA-PFP and d3Me-AA-PFP derivatives of AA are readily extractable into water-immiscible, GC-compatible organic solvents such as toluene. d0Me-AA-PFP and d3Me-AA-PFP derivatives are stable in toluene extracts for several weeks, thus enabling high throughput quantitative measurement of biological AA by GC-MS using in situ prepared d3Me-AA as internal standards in OMICS format. Here, we describe the development of a novel OMICS-compatible QC system and demonstrate its utility for the quality control of quantitative analysis of 21 free AA and metabolites in human plasma samples by GC-MS as Me-PFP derivatives. The QC system involves cross-standardization of the concentrations of the AA in their aqueous solutions at four concentration levels and a quantitative control of AA at the same four concentration levels in pooled human plasma samples. The retention time (tR)-based isotope effects (IE) and the difference (δ(H/D) of the retention times of the d0Me-AA-PFP derivatives (tR(H)) and the d3Me-AA-PFP derivatives (tR(D)) were determined in study human plasma samples of a nutritional study (n = 353) and in co-processed QC human plasma samples (n = 64). In total, more than 400 plasma samples were measured in eight runs in seven working days performed by a single person. The proposed QC system provides information about the quantitative performance of the GC-MS analysis of AA in human plasma. IE, δ(H/D) and a massive drop of the peak area values of the d3Me-AA-PFP derivatives may be suitable as additional parameters of qualitative analysis in targeted GC-MS amino acid-OMICS.

GC-MS/MS Determination of Synthetic Cathinones: 4-chloromethcathinone, N-ethyl Pentedrone, and N-ethyl Hexedrone in Oral Fluid and Sweat of Consumers under Controlled Administration: Pilot Study.

This study presents a validated GC-MS/MS method for the detection and quantification of 4-chloromethcathinone or clephedrone (4-CMC), N-ethyl Pentedrone (NEP), and N-ethyl Hexedrone (NEH, also named HEXEN) in oral fluid and sweat and verifies its feasibility in determining human oral fluid concentrations and pharmacokinetics following the administration of 100 mg of 4-CMC orally and 30 mg of NEP and NEH intranasally. A total of 48 oral fluid and 12 sweat samples were collected from six consumers. After the addition of 5 μL of methylone-d3 and 200 μL of 0.5 M ammonium hydrogen carbonate, an L/L extraction was carried out using ethyl acetate. The samples, dried under a nitrogen flow, were then derivatized with pentafluoropropionic anhydride and dried again. One microliter of the sample reconstituted in 50 μL of ethyl acetate was injected into GC-MS/MS. The method was fully validated according to international guidelines. Our results showed how, in oral fluid, the two cathinones taken intranasally were absorbed very rapidly, within the first hour, when compared with the 4-CMC which reached its maximum concentration peak in the first three hours. We observed that these cathinones were excreted in sweat in an amount equivalent to approximately 0.3% of the administered dose for 4-CMC and NEP. The total NEH excreted in sweat 4 h after administration was approximately 0.2% of the administered dose. Our results provide, for the first time, preliminary information about the disposition of these synthetic cathinones in the consumers' oral fluid and sweat after controlled administration.

Ultrafast Charging of a 4.8 V Manganese‐Rich Cathode‐Based Lithium Metal Cell by Constructing Robust Solid Electrolyte Interphases

AbstractFast charging of Li‐metal battery (LMB) is a challenging issue owing to the interfacial instability of Li‐metal anode in liquid electrolyte and Li‐dendrites growth, resulting in fire hazard. Those issues motivated to pioneer a stabilization strategy of liquid electrolyte‐derived solid electrolyte interphase (SEI) layer that enables dendrites‐free Li‐metal anode under extremely high current density, which solid‐state battery cannot. Here, the novel electrolyte formulation is reported including trace‐level pentafluoropropionic anhydride (PFPA) combined with fluoroethylene carbonate (FEC) additives, and the SEI stabilization in Li//Mn‐rich LMB, achieving unprecedented ultrafast charging under simultaneous extreme conditions of 20 C (charged in 3 min), 4.8 V and 45 °C, delivering 118 mAh g−1 for long reversible 400 cycles, and unprecedented high stability of Li//Li cell under extremely high current 10 mA cm−2 (Li stripping/plating in 6 min) for a prolonged time of 200 h. The SEI analysis results reveal that the PFPA, which has a symmetric 10 F‐containing molecular structure, is a strong F source for promptly producing thin, uniform, and robust F‐ and organics‐enriched SEI layers at both Li‐metal anode and Mn‐rich cathode, preventing Li‐dendrites. This study provides a potential concept for ultrafast charging, long‐cycled, and safer high‐energy LMBs and LIBs.

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.

Nanoconfinement effect based in-fiber extraction and derivatization method for ultrafast analysis of twenty amines in human urine by GC-MS: Application to cancer diagnosis biomarkers’ screening

Amines, including amino acids and biogenic amines are widely recognized as cancer biomarkers for early diagnosis and treatment. But rapid analysis of amines in biological samples with high sensitivity, good durability and low cost is still challenging. Herein, we proposed an ultrafast biological sample preparation method based on in-fiber extraction and derivatization using a homemade needle-tip devise. Under the nanoconfinement effect, the extraction was completed within 2 min in nanofibers, followed by the in-suit derivatization within 3 min under room temperature using pentafluoropropionic anhydride (PFA) as derivatization reagent, then the needle was inserted into the inlet of GC-MS directly for thermal desorption to enable the complete injection. Additionally, the established sample preparation strategy consumed only 20 μL organic solvent and urine sample, and the sensitivity was increased compared with other methods. After systematical optimization and validation, coupled to GC-MS, the established method was successfully applied to quantify twenty amines in urine of healthy people and lung, colorectal and breast cancer patients. The diagnosis biomarkers for each cancer were screened out and accessed by multiple statistical approaches. Then we further constructed the cancer diagnosis model of each cancer through binary logistic regression analysis for clinical use. Taken together, the established nanoconfinement effect based in-fiber extraction and derivatization biological sample preparation strategy in combination with GC-MS was an ultrafast method for biological analysis with high sensitivity, good durability, little organic solvent consumption and low cost. It possesses great potential in clinical cancer screening and treatment.

Development, validation of a GC–MS method for the simultaneous measurement of amino acids, their PTM metabolites and AGEs in human urine, and application to the bi-ethnic ASOS study with special emphasis to lysine

A gas chromatography-mass spectrometry (GC–MS) method was developed and validated in relevant concentration ranges for the simultaneous measurement of l-lysine (Lys, L) and its Nε- and Nα-methylated (M), Nε- and Nα-acetylated (Ac), Nε-carboxymethylated (CM) and Nε-carboxyethylated (CE) metabolites in human urine. Analyzed Lys metabolites were the post-translational modification (PTM) products Nε-mono-, di- and trimethyllsine, Nε-MML, Nε-DML, Nε-TML, respectively, Nα-ML, Nε-AcL, Nα-AcL, and its advanced glycation end-products (AGEs) Nε-CML, Nε-CM-[2,4,4-2H3]Lys (d3-CML), Nε-CEL and furosine. AGEs of arginine (Arg) and cysteine (Cys) were also analyzed. De novo synthesized trideutero-methyl esters (R-COOCD3) from unlabelled amino acids and derivatives were used as internal standards. Native urine samples (10 µL aliquots) were evaporated to dryness under a stream of nitrogen. Analytes were esterified using 2 M HCl in methanol (60 min, 80 °C) and subsequently amidated by pentafluoropropionic anhydride in ethyl acetate (30 min, 65 °C). The generated methyl ester-pentafluoropropionyl (Me-PFP) derivatives were reconstituted in borate buffer and extracted immediately with toluene. GC–MS analyses were performed by split-less injection of 1-µL aliquots, oven-programmed separation and negative-ion chemical ionization (NICI). Mass spectra were generated in the scan mode (range, m/z 50–1000). Quantification was performed in the selected-ion monitoring (SIM) mode using a dwell time of 50 or 100 ms for each ion. The GC–MS method was suitable for the measurement of Lys and all of its metabolites, except for the quaternary ammonium cation Nε-TML. The Me-PFP derivatives of Lys, Arg and Cys and its metabolites eluted in the retention time window of 9 to 14 min. The derivatization of Nε-CML, d3-CML and Nε-CEL was accompanied by partial Nε-decarboxylation and formation of the Me-PFP Lys derivative. The lowest derivatization yield was observed for Nε-DML, indicating a major role of the Nε-DML group in Lys derivatization. The GC–MS method enables precise (relative standard deviation, RSD < 20%) and accurate (bias, < ± 20%) simultaneous measurement of 33 analytes in human urine in relevant concentration ranges. We used the method to measure the urinary excretion rates of Lys and its PTM metabolites and AGEs in healthy black (n = 39) and white (n = 41) boys of the Arterial Stiffness in Offspring Study (ASOS). No remarkable differences were found indicating no ethnic-related differences in PTM metabolites and AGEs except for Nε-monomethyllysine and S-(2-carboxymethylcysteine).

A multi-analytical study of historical coated plaster surfaces: the examination of a nineteenth-century V&A cast of a tombstone

A multi-analytical study was designed to characterise historical coated plaster surfaces. The method was applied to investigate the surface coatings of the nineteenth-century plaster cast of the tombstone of the Presbyter Bruno that belongs to the Victoria and Albert Museum collection. At first, selected samples of the object were examined with Visible Light Reflectance and Ultra-Violet Fluorescence Optical Microscopy (VLR- and UVf-OM respectively) and Scanning Electron Microscopy (SEM) demonstrating a consistent stratigraphy featuring a bulk, an interface and an uppermost layer. The latter layer appeared to consist of an aged coating and dirt. Overpainted and repaired areas of the object generated samples that had additional layers on top of the aforementioned stratigraphy. A layer that seemed to be an additional surface varnish or a coating that had not been absorbed to the bulk has been observed in a couple of samples. Elemental characterization was carried out with energy dispersive X-ray spectroscopy (EDS) and further analyses were performed with X-ray diffraction (XRD) and Fourier-transform infrared (FT-IR) spectroscopy with focal plane array (FPA) imaging which confirmed that the bulk of the object is made of gypsum plaster containing mostly silicate and carbonate inclusions. Gas chromatography/mass spectrometry (GC/MS) and pyrolysis-GC/MS with extraction methods based on n-propanol followed by pentafluoropropionic anhydride (PFPA), tetramethylammonium hydroxide (TMAH) and 3-trifluoromethylphenyltrimethylammonium hydroxide (m-TFPTAH) were performed to detect organic media. The results suggest that the organic medium used for the surface coating is a diterpenic resin that contained silicon, aluminium and traces of other inorganic elements. The organic medium of overpainted areas was based on alkyd resins and the in-paints were characterised as a blend of silicon and barium at varied concentrations. This multi-analytical approach can generate a better understanding of manufacturing, component materials and conservation issues of coated plaster objects.

GC-MS Studies on Derivatization of Creatinine and Creatine by BSTFA and Their Measurement in Human Urine.

In consideration of its relatively constant urinary excretion rate, creatinine (2-amino-1-methyl-5H-imidazol-4-one, MW 113.1) in urine is a useful endogenous biochemical parameter to correct the urinary excretion rate of numerous endogenous and exogenous substances. Reliable measurement of creatinine by gas chromatography (GC)-based methods requires derivatization of its amine and keto groups. Creatinine exists in equilibrium with its open form creatine (methylguanidoacetic acid, MW 131.1), which has a guanidine and a carboxylic group. Trimethylsilylation and trifluoroacetylation of creatinine and creatine are the oldest reported derivatization methods for their GC-mass spectrometry (MS) analysis in human serum using flame- or electron-ionization. We performed GC-MS studies on the derivatization of creatinine (d0-creatinine), [methylo-2H3]creatinine (d3-creatinine, internal standard) and creatine (d0-creatine) with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) using standard derivatization conditions (60 min, 60 °C), yet in the absence of any base. Reaction products were characterized both in the negative-ion chemical ionization (NICI) and in the positive-ion chemical ionization (PICI) mode. Creatinine and creatine reacted with BSTFA to form several derivatives. Their early eluting N,N,O-tris(trimethylsilyl) derivatives (8.9 min) were found to be useful for the precise and accurate measurement of the sum of creatinine and creatine in human urine (10 µL, up to 20 mM) by selected-ion monitoring (SIM) of m/z 271 (d0-creatinine/d0-creatine) and m/z 274 (d3-creatinine) in the NICI mode. In the PICI mode, SIM of m/z 256, m/z 259, m/z 272 and m/z 275 was performed. BSTFA derivatization of d0-creatine from a freshly prepared solution in distilled water resulted in formation of two lMate-eluting derivatives (14.08 min, 14.72 min), presumably creatinyl-creatinine, with the creatininyl residue existing in its enol form (14.08 min) and keto form (14.72 min). Our results suggest that BSTFA derivatization does not allow specific analysis of creatine and creatinine by GC-MS. Preliminary analyses suggest that pentafluoropropionic anhydride (PFPA) is also not useful for the measurement of creatinine in the presence of creatine. Both BSTFA and PFPA facilitate the conversion of creatine to creatinine. Specific measurement of creatinine in urine is possible by using pentafluorobenzyl bromide in aqueous acetone.

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.

Measurement of the tripeptides glutathione and ophthalmic acid by gas chromatography-mass spectrometry

Glutathione (GSH, γ-glutamyl-cysteinyl-glycine) is the most abundant intracellular dicarboxylic tripeptide with multiple physiological roles. GSH and its symmetric disulfide GSSG co-exist in biological samples. The GSH-to-GSSG molar ratio is high within cells and low in extracellular compartments. It is widely used as a biomarker of oxidative stress. Here, we report on a novel two-step derivatization procedure which allows for the specific quantitative measurement of GSH by gas chromatography-mass spectrometry (GC-MS) in the electron-capture negative-ion chemical ionization mode. The method is based on the derivatization of GSH first with pentafluoropropionic anhydride in ethyl acetate (30 min, 65 °C) and subsequently with 2 M HCl/CH3OH (30 min, 80 °C). Esterification in 2 M HCl/CD3OD is used for the in situ synthesis of deuterium-labelled GSH for use as internal standard. Quantification is performed by selected-ion monitoring of m/z 557 for GSH and m/z 560 for the internal standard (1 mM). Linear regression analysis between the peak area ratio of m/z 557 to m/z 560 (y) and the GSH concentration (x; range, 0–1 mM) resulted in the regression equations y = 0.02 + 0.62x (r2 = 0.976) in the absence and y = 0.02 + 0.63x (r2 = 0.997) in the presence of GSSG (range, 0–1 mM), underlying the linearity of the method for GSH and suggesting lack of interference by GSSG. γ-Glu-Cys and Cys-Gly also did not interfere. The GC-MS method demonstrated that in human hemolysate, nitrite at toxicological concentrations (range, 0–15 mM) depletes erythrocytic GSH. For the GC-MS analysis of the GSH tripeptide analogue ophthalmic acid, which lacks a Cys moiety, the derivatization order was HCl/CH3OH followed by pentafluoropropionic anhydride in ethyl acetate.

Activity-based ratiometric fluorescent small-molecule probe for endogenously monitoring neutrophil elastase in living cells

Neutrophil elastase (NE), a representative protease which is closely related to many diseases, acts an indispensable role in inflammatory diseases and clinical medicine. In this work, one activity-based non-peptide ratiometric fluorescent probe DCDF was designed with pentafluoropropionic anhydride as identification group. To our knowledge, this is the first probe capable of detecting NE in ratio. After the addition of the NE, the emission spectrum of DCDF has obvious bathochromic-shift phenomenon, and there is large Stokes shifts of ∼60 nm. Compared to only a few reported NE probes, DCDF is sensitive and selective and has very low detection limit (0–14 μg/mL, DL = 30.8 ng/mL). A possible response mechanism was proposed and verified by HPLC and HRMS spectra. What’s more, DCDF is capable of endogenous recognition imaging in biological cells without interference from other enzymes under the ratio signal. A549 and HeLa cells were used for endogenous cell imaging experiments of NE and the feasibility of DCDF for the specific detection of NE in cells was proved. This experimental result makes probe DCDF a very promising tool for the clinical diagnosis and treatment of NE related diseases.

The effects of fluoroalkyl chain length and density on siRNA delivery of bioreducible poly(amido amine)s

Fluorination is an attractive strategy for the improvement of transfection efficiency of nucleic acid delivery vectors. Bioreducible poly(amido amine)s (bPAAs) are an important class of biomaterials exhibited to effectively deliver multiple nucleic acids. However, still, the effects of fluoroalkyl chain length and density of bPAA on siRNA delivery are unveiled. Here, we synthesized bPAAs and grafted with different chain lengths and densities of fluorocarbon compounds. Furthermore, we prepared a library of complexes of fluorinated bPAA and siRNA, and investigated the effects of fluorination on the siRNA delivery in vitro and in vivo. We found that all the synthesized bPAAs readily formed complexes with siRNA and the fluorinated complexes considerably achieved improved gene silencing efficacies both in vitro and in vivo. Dramatically, the gene silencing efficacy was increased with increasing fluorine contents. Heptafluorobutyric anhydride (HF) modified bPAAs achieved better gene silencing efficacy when compared with bPAAs fluorinated by trifluoroacetic anhydride (TF) and pentafluoropropionic anhydride (PF) providing the evidence for choosing of best one among fluorocarbon compounds. In addition, a combination of fluorination with bioreducibility enables efficient and safe siRNA delivery.

Hybrid Solid-Phase Extraction for Selective Determination of Methamphetamine and Amphetamine in Dyed Hair by Using Gas Chromatography–Mass Spectrometry

Sample preparation is an important step in the isolation of target compounds from complex matrices to perform their reliable and accurate analysis. Hair samples are commonly pulverized or processed as fine cut, depending on preference, before extraction by techniques such as solid-phase extraction (SPE), liquid–liquid extraction, and other methods. In this study, a method based on hybrid solid-phase extraction (hybridSPE) and gas chromatography–mass spectrometry (GC–MS) was developed and validated for the determination of methamphetamine (MA) and amphetamine (AP) in hair. The hair samples were mechanically pulverized after washing with de-ionized water and acetone. The samples were then sonicated in methanol at 50 °C for 1 h and centrifuged at 50,000× g for 3 min. The supernatants were transferred onto the hybridSPE cartridge and extracted using 1 mL of 0.05 M methanolic hydrogen chloride. The combined solutions were evaporated to dryness, derivatized using pentafluoropropionic anhydride, and analyzed by GC–MS. Excellent linearity (R2 > 0.9998) was achieved in the ranges of 0.05–5.0 ng/mg for AP and 0.1–10.0 ng/mg for MA. The recovery was 83.4–96.8%. The intra- and inter-day accuracies were −9.4% to 5.5% and −5.1% to 3.1%, while the intra- and inter-day precisions were within 8.3% and 6.7%, respectively. The limits of detections were 0.016 ng/mg for AP and 0.031 ng/mg for MA. The validated hybridSPE method was applied to dyed hair for MA and AP extraction and compared to a methanol extraction method currently being used in our laboratory. The results showed that an additional hybridSPE step improved the recovery by 5.7% for low-concentration quality control (QC) samples and by 24.1% for high-concentration QC samples. Additionally, the hybridSPE method was compared to polymeric reversed-phase SPE methods, and the absolute recoveries for hybridSPE were 50% and 20% greater for AP (1.5 ng/mg) and MA (3.0 ng/mg), respectively. In short, the hybridSPE technique was shown to minimize the matrix effects, improving GC–MS analysis of hair. Based on the results, the proposed method proved to be effective for the selective determination of MA and AP in hair samples.

Effect of long- and short-chain perfluorinated compounds on cultured thyroid cells viability and response to TSH.

Perfluorinated chemicals are widespread pollutants persistent in the environment with links to some major health issues. The two main compounds, perfluoro-octanoic acid (PFOA) and perfluoro-alkyl sulphonate (PFOS), were recently classified as carcinogenetic and thus their use has been restricted. Short-chain PFCs were recently developed as an alternative, but no data regarding the possible endocrine toxicities of these compounds are available. Aim of this study was to investigate whether short-chain PFCs could jeopardize thyroid cell viability and/or interfere with the functional effect TSH. Fisher rat thyroid line-5 (FRTL-5) was treated with increasing concentrations of PFOA, PFOS, perfluorobutanesulfonic acid (PFBS), perfluorobutanoic acid (PFBA), pentafluoropropionic anhydride (PFPA), perfluoropentanoic acid (PFPeA) to evaluate modifications in cell viability and TSH-stimulated cAMP production. Neither long nor short-chain PFCs affected cell viability (apart from PFOS 100µM), or interfered with cAMP production. The results of the present study demonstrate for the first time that short-chain PFCs have no acute cytotoxic effect on thyroid cells in vitro and that cAMP production is not modulated by any of the tested PFCs.

Production of AEME in hair after thermal hair treatment

Several studies have shown that thermal straightening has an influence on the concentration of THC, ethyl glucuronide and cocaine in hair. Heat treatment of hair may decrease the concentration of cocaine in hair and increase BZE and cocaethylene. A BZE/cocaine ratio greater than 1 was proposed to identify thermal straightening prior to sampling [1] . The aim of this study was to evaluate the influence of thermal straightening on other metabolites of cocaine: ecgonine methyl ester (EME), norcocaine and on the marker of crack use: anhydroegonine methyl ester (AEME). Forty-one capillary samples positive for cocaine were treated in vitro with iron plates heated to 200 °C. One lock of hair was put in contact 30 times for 2 seconds with a hair straightener, the other lock was not treated. After decontamination and pulverization, both hair locks were incubated in a phosphate buffer for 2 h in an ultrasonic bath. Analytes with their deuterated analogues were isolated using solid phase extraction and then derivatized with pentafluoropropionic anhydride and pentafluoropropanol. The derivatized products were determined by GC-MS in EI mode. Hair concentrations of cocaine, BZE, EME, cocaethylene, norcocaine and AEME were compared with those in the corresponding untreated strands. After treatment, a median decrease of concentrations was observed for cocaine (−55.0%), EME (−28.2%), cocaethylene (−41.6%) and norcocaine (−43.2%), whereas a median increase was found for BZE (+28.6%) and AEME (+122%). The decreases of cocaine, EME, cocaethylene and norcocaine concentrations after treatment with a heating iron are most likely due to thermal denaturation of the mentioned substances. The increased concentrations of BZE and AEME could be explained by chemical transformation of cocaine which is incorporated in the hair matrix into BZE and AEME. Our results show that presence of AEME in hair does not prove that crack was consumed. A crack use should only be considered in case where thermal straightening before sampling may be excluded (BZE/cocaine ratio lower 1). Regarding our in vitro results, thermal straightening should be documented during hair collection and should be considered for interpretation of AEME results in hair.

Development and validation of GC-MS methods for the comprehensive analysis of amino acids in plasma and urine and applications to the HELLP syndrome and pediatric kidney transplantation: evidence of altered methylation, transamidination, and arginase activity.

We developed and validated gas chromatography-mass spectrometry (GC-MS) methods for the simultaneous measurement of amino acids and their metabolites in 10-µL aliquots of human plasma and urine. De novo synthesized trideutero-methyl esters were used as internal standards. Plasma proteins were precipitated by acidified methanol and removed by centrifugation. Supernatants and native urine were evaporated to dryness. Amino acids were first esterified using 2M HCl in methanol and then amidated using pentafluoropropionic anhydride for electron-capture negative-ion chemical ionization. Time programmes were used for the gas chromatograph oven and the selected-ion monitoring of specific anions. The GC-MS methods were applied in clinical studies on the HELLP syndrome and pediatric kidney transplantation (KTx) focusing on L-arginine-related pathways. We found lower sarcosine (N-methylglycine) and higher asymmetric dimethylarginine (ADMA) plasma concentrations in HELLP syndrome women (n = 7) compared to healthy pregnant women (n = 5) indicating altered methylation. In plasma of pediatric KTx patients, lower guanidinoacetate and homoarginine concentrations were found in plasma but not in urine samples of patients treated with standard mycophenolate mofetil-based immunosuppression (MMF; n = 22) in comparison to matched patients treated with MMF-free immunosuppression (n = 22). On average, the global arginine bioavailability ratio was by about 40% lower in the MMF group compared to the EVR group (P = 0.004). Mycophenolate, the major pharmacologically active metabolite of MMF, is likely to inhibit the arginine:glycine amidinotransferase (AGAT), and to enhance arginase activity in leukocytes and other types of cell of MMF-treated children.

Pentafluoropropionic Anhydride Functionalized PAMAM Dendrimer as miRNA Delivery Reagent

Poly(amidoamine) (PAMAM) dendrimers are good candidates for nucleic acid delivery with their well-defined characteristics. MicroRNA (miRNA) mediated regulation of biological process are also active area of investigation. Fibroblast cells, such as MRC-5, are one of the cell lines used in biological researches due to their hard to transfect nature. In this two-staged study, cystamine core G5 PAMAM dendrimers were synthesized and modified with pentafluoropropionic anhydride (PA) and subsequently tested as miRNA delivery reagent on MRC-5 cells. Effect of fluorination against to naked G5 dendrimer over transfection efficiency was also investigated by molecular docking and quantitative structure-activity relationship (QSAR) calculations. Structural characterization of the synthesized dendrimers was verified by spectroscopic techniques. Gel retardation assay, particle size and transmission electron microscopy results demonstrated polyplex formation ability of fluorinated dendrimers with miRNA at nanoscale level. Zeta potential values indicated non-aggregation and increased stability of the polyplexes. Prepared polyplexes with fluorinated dendrimer showed over 90% cell viability and transfection efficiency. In silico calculations confirmed the stable complexation with miRNA and smooth penetration capability into the cell.