Picolinic acid

HPLC grade water and acetonitrile (ACN) were purchased from (VWR International-UK). Acetone was purchased from (Fisher Scientific, Loughborough, UK). HPLC grade methanol (MeOH, ≥99.9%), HPLC grade chloroform (CHCl₃, ≥99.9%), 5-chloro-2-mercaptobenzothiazole (5C2M), 2-mercaptobenzothiazole, 6-aza-2-thiothymine, 2-(4-hydroxyphenylazo) benzoic acid, 2,5-dihydroxy benzoic acid (DHB), super-DHB, sinapic acid, 2,4,6-trihydroxy acetophenone monohydrate, 2,5-dihydroxy acetophenone, α-cyano-4-hydroxycinnamic acid, picolinic acid, and 9-aminoacridine hemihydrate were purchased from (Sigma Aldrich, Gillingham, UK). Porcine Gb3 standard and N-heptadecanoyl ceramide trihexoside (C17:0) internal standard were purchased from Matreya (Pleasant Gap, PA, USA).

Picolinic acid was purchased
from Sigma-Aldrich and used without further purification. Pterostilbene
was purchased from Dynveo and purified following the procedure described
in ref (11 (link)). Single
crystals suitable for an SXCRD analysis were obtained as follows.
Polymorph A: a pterostilbene/Picolinic acid cocrystal (polymorph A)
(20 mg, 0.053 mmol) was dissolved in toluene (0.3 mL) at 65 °C.
Then, the solution was cooled to 25 °C and stored sealed. Single
crystals were observed after 145 days. Polymorph B: pterostilbene
(50 mg, 0.195 mmol) and Picolinic acid (24 mg, 0.195 mmol) were dissolved
together in methyl ethyl acetone (0.4 mL) at 25 °C and stored
sealed. Single crystals were observed after 289 days.

Normal standards—tryptophan, l-kynurenine, pyridine-2,3-dicarboxylic acid (QUIN), KYNA, picolinic acid, 3-HK—were purchased from Sigma-Aldrich (MO, USA). The Internal standards (IS)—tryptophan-d₃, l-kynurenine-d₄, QUIN-d₃ [13C6], 3-HK-d₃—were purchased from Toronto Research Chemicals Canada (Toronto Canada). KYNA-d₅ and picolinic acid-d₄ were obtained from C/D/N Isotopes Inc. (Quebec, Canada). Solutions for the mobile phases—water, methanol and formic acid 99%—were all LC-MS grade from Chromasolve, Honeywell, VWR International AB, Stockholm (Sweden). Solutions for plasma preparations: ammonia (32%) was purchased from VWR and ZnSO₄ was purchased from Sigma-Aldrich (MO, USA). Stock solutions of all unlabeled standards (tryptophan, kynurenine, QUIN, KYNA, picolinic acid, and 3-HK) were prepared in water for HPLC, LCMS grade, and stored at – 20 °C. Calibrators were generated mixing all compounds in a final solution of 8.3 μM and 10 times higher for tryptophan, 83 μM.

Tryptophan, kynurenine, kynurenic acid, anthranilic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid, xanthurenic acid, picolinic acid, and quinolinic acid were obtained from Sigma Aldrich (Poole, United Kingdom). The stable isotope–labeled internal standards were ²H₅-Tryptophan (QMx Laboratories, Thaxted, United Kingdom), ²H₆-kynurenine sulfate and ²H₅-kynurenic acid (CK isotopes, Ibstock, United Kingdom), and ²H₃-picolinic acid and ¹³C₃,¹⁵N-quinolinic acid (LGC standards, Teddington, United Kingdom). Acetonitrile and methanol were obtained from Rathburn Chemicals (Walkerburn, United Kingdom) and Fisher Scientific UK (Loughborough, United Kingdom), respectively.
Standards of each analyte were used to automatically tune in multiple reaction monitoring (MRM) mode. Transition optimizations were performed manually. xanthurenic acid was significantly more sensitive in negative ion mode, but, with the chromatographic conditions used, there was massive matrix ion suppression. Consequently, xanthurenic acid was measured in positive ion mode.
Because analyte retention times on Chirobiotic-T columns tend to be matrix dependent, there is a critical requirement for the appropriate stable isotope internal standards. Consequently, when stable isotopes were unavailable, 2 separate transitions, when possible, were used to calculate and then confirm the result.