Phenylisothiocyanate pitc

Theobromine, pentoxifylline, propentofylline, HPLC-grade pyridine, phenyl isothiocyanate (PITC) and ammonium acetate were purchased from Merck (Darmstadt, Germany), whereas caffeine, theophylline and all other chemicals including high performance liquid chromatography (HPLC)-grade water, ethanol, and methanol were purchased from Fisher Scientific (Schwerte, Germany). The following standards from Avanti Polar Lipids were used for normalization: 06:0 PC (DHPC), 19:0 Lyso PC, 06:0 SM (d18:1/6:0), Cer d18:1/6:0, D7-cholesterol and Splash^® II Lipidomix^® Mass Spec Internal Standard.

Methanol (HPLC gradient grade), ethanol, and tetrachloroethylene (synthesis grade) were purchased from Scharlab (Barcelona, Spain). CuSO₄·7H₂O, phenylisothiocyanate (PITC), 2,4,6-tri(2-pyridyl)-triazine (TPTZ), neocuproine, and DPPH were supplied by Sigma-Aldrich (St. Louis, MO, USA). Triethylamine (TEA), hydrochloric acid (37%), sodium acetate, glacial acetic acid, Fe₃Cl·6H₂O and FeSO₄·7H₂O, trichloroacetic acid (TCA), butylated hydroxytoluene (BHT), and 2-thiobarbituric acid (TBA) (analysis quality) were supplied by Panreac (Barcelona, Spain). Formic acid (synthesis grade) and amino acid standards Glu, His, Met, arginine (Arg), aspartic acid (Asp), lysine (Lys), proline (Pro), valine (Val), isoleucine (Ile), and phenylalanine (Phe) were provided by Merck (Darmstadt, Germany). Polyphenol standards were supplied as follows: gallic acid (GAL), protocatechuic acid (PCA), p-coumaric acid (COU), ferulic acid (FA), catechin (CAT), vanillic acid (VAN), epicatechin (ECAT), syringic acid (SYR), and Trolox (TR) by Sigma-Aldrich Chemie (Steinheim, Germany); rutin (RU) and gentisic acid (GA) by Merck (Darmstadt, Germany). Ultrapure water was obtained from a Milli-Q system from Millipore (Bedford, MA, USA).

α-Methyl-DL-tryptophan (α-Me-DL-Trp, crystalline), L-tryptophan (Trp, 99.5%), amino acid analytical standard, L-norleucine (NorLeu, 98%), and phenyl isothiocyanate (PITC, ≥99%) as well as all other solvents and chemicals were of analytical quality and were purchased from Sigma-Aldrich (Buchs, Switzerland). Bovine serum albumin (BSA; 97%), was bought from VWR International GmbH (Dietikon, Switzerland). Water of Milli-Q quality (Merck Millipore) from Merck KGaA (Darmstadt, Germany) was used for all experiments. All mixtures of liquids given as ratios refer to volume to volume ratios (v/v) unless otherwise noted.

Seven commercial cultivars of seed‐rich pumpkin in China, including Dali, Luoren No.2, Luoren No.3, Jinhui No. 4, Ruifeng No.9, Yinhui No. 3, and Xuecheng No.2 (coded LR‐1, LR‐2, LR‐3, JH‐4, RF‐9, YH‐3, and XC‐2), were purchased from Mudanjiang Beidahuang Food Co., Ltd. All seeds were received in good shape (without damage) and were stored at 4℃.
Gamma‐aminobutyric acid (GABA) and phenylisothiocyanate (PITC) were purchased from Sigma Chemical Co. Monosodium glutamate (MSG), trimethylamine, n‐hexane, methyl alcohol, acetonitrile of HPLC grade, and other analytical reagents were obtained from Sinopharm Chemical Reagent Co., Ltd.

Sorghum grains cultivar (Butanna) was obtained from the Arab‐Sudanese seed company, Khartoum, Sudan. The materials were carefully cleaned and stored in polythene bags at 4°C before use. GABA, PLP, and phenylisothiocyanate (PITC) were obtained from Sigma Chemicals Co. Acetonitrile was of high‐performance liquid chromatography (HPLC) grade. All other chemicals and reagents used were of analytical grade.

The analytical standards of 20 amino acids, including aspartic acid (Asp), glutamic acid (Glu), γ-am aminobutyric acid (GAMA), serine (Ser), glutamine (Gln), histidine (His), asparagine (Asn), glycine (Gly), threonine (Thr), arginine (Arg), alanine (Ala), tyrosine (Tyr), valine (Val), methionine (Met), tryptophan (Trp), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), lysine (Lys), and proline (Pro), were obtained from Sigma-Aldrich Co. (St. Louis, Mo, USA) with all purity ≥ 99%. Phenyl isothiocyanate (PITC) (purity ≥ 99%) and triethylamine (purity ≥ 99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ammonium acetate acetonitrile (HPLC grade) and hexane (HPLC grade) were obtained from Dikma (Beijing, China). Ultra-pure water was prepared using a Milli-Q plus system (Millipore, Boston, Massachusetts, The United States of America).
The individual stock solutions were prepared in 0.1 mol/L HCl solution at the concentration of 10,000 μg/mL. The standard mixture was prepared from the individual stock standard solutions at the concentration of 100 μg/mL. The working solutions of the amino acid were prepared by serial dilutions. The internal standard norleucine was prepared with a concentration of 100 μg/mL. All the solutions were stored at 4 °C in the refrigerator.