Menaquinone 4 mk 4

The following chemicals were used: citric acid (analytical grade) was from Serva Feinbiochemica GmbH, Germany, trisodium citrate pentahydrate (extra pure) was from Laborchemie, Apolda GmbH, Germany, disodium monohydrogen phosphate dihydrate (Na₂HPO₄·2H₂O) (≥ 98%), sodium hydroxide (NaOH) (≥ 99%), potassium chloride (KCl) (≥ 99.5%), chloroform (HPLC grade), and methanol (≥ 99.98%, ultra LC–MS grade) were from Carl Roth GmbH, Germany, monosodium dihydrogen phosphate dihydrate (NaH₂PO₄·2H₂O) (pure pharma grade) was from Applichem GmbH, Germany, disodium carbonate monohydrate (Na₂CO₃·H₂O) (> 99%) was from Fluka Chemika, Germany, sodium bicarbonate (NaHCO₃) was from Merck, Germany, DMPC (14:0 PC) (1,2-dimyristoyl-sn-glycero-3-phosphocholine) lipid was from Avanti Polar Lipids, USA, menaquinone 4 (MK-4) and menaquinone 7 (MK-7) were from Sigma Aldrich, Germany; menaquinone 9 (MK-9) was from Caymann Chemical, Germany. The buffer solutions were prepared using citric acid/trisodium citrate pentahydrate for pH 4, Na₂HPO₄·2H₂O/NaH₂PO₄·2H₂O for pH 6 and 7.4, Na₂CO₃·H₂O/NaHCO₃ for pH 9, Na₂HPO₄·2H₂O/NaOH for pH 11 and 11.4 and NaOH for pH 12–14.

Tissues used in this study were collected from 1-year-old (leaves, stems, bark, and roots) and mature elite (flowers and hulls) black walnut trees located at Martell Forest (West Lafayette, IN, USA). Naphthoquinone standards of juglone, phylloquinone, menaquinone-4 (MK-4), and plumbagin were from Sigma-Aldrich. Unless otherwise mentioned, all other reagents were from Fisher Scientific. Ultrapure water and high-performance liquid chromatographic (HPLC)- or gas chromatographic (GC)-grade solvents were used for all metabolite extractions. HPLC analyses were carried out on an Agilent 1260 Infinity series instrument (Agilent Technologies) equipped with diode array and fluorometer detection modules employing Chemstation software. An Agilent 7890B GC coupled with a 5977A mass spectrometer (MS) employing Chemstation software was used to perform GC-MS analyses.