Biomedical Chromatography

Bhushan, R.; Joshi, Shalini

doi: 10.1002/bmc.1130070502pmid: 8305853

Application of HPLC as a prime tool in the area of enantiomeric resolution has opened doors of success and varied interest. Use of chiral reagents either indirectly (as derivatization reagent) or directly (added to stationary or mobile phase) has led to achieve resolution of a wide range of compounds. Amino acids, being important molecules with simple structure and easy availability, have been extensively studied. A bibliographic survey on HPLC resolution of amino acids and derivatives along with a brief discussion on general methods of enantiomeric separation has been presented.

Qureshi, G. Ali; Bednar, Ivan; Min, Qian; Södersten, Per; Silberring, Jerzy; Nyberg, Fred; Thörnwall, Madeleine

doi: 10.1002/bmc.1130070503pmid: 8305854

A sensitive HPLC method has been described for quantitation of two cholecystokinin (CCK) peptides in discrete rat brain regions. Separation and quantitation was performed by the reversed‐phase HPLC combined with electrochemical detection. Analytical recoveries of the tetrapeptide (CCK‐4) and octapeptide‐sulphate (CCK‐8s) were 96% and 94%, respectively. The between assay coefficient of variation (CV) was less than 3% for both peptides. The within‐assay CV was 4% and 6% for CCK‐4 and CCK‐8s and the detection limit was 2 and 10 pmol/mL, respectively. For identification of structures, the peptides were fractionated by semi‐preparative HPLC using a novel SMART system for micropurification. The fractions were analysed by fast atom bombardment mass spectrometry (FAB MS) which confirmed the presence of both CCK‐4 and CCK‐8s in the rat brain tissue.

Chaga, Grigoriy; Porath, Jerker; Illéni, Tibor

doi: 10.1002/bmc.1130070504pmid: 8305855

Amyloglucosidase from Halobacterium sodomense was purified by a combination of hydrophobic interaction chromatography and immobilized metal ion affinity chromatography at analytical and preparative scale with 75% recovery. The enzyme was found to be a dimer of two different subunits with molecular weights of 72,000 and 82,000 D, respectively, combining in a 175,000 D native protein. The specific activity, KM, and amino acid composition of the enzyme was determined.

Hayakawa, Kazuichi; Terai, Noriko; Suzuki, Kumiko; Dinning, Paul G.; Yamada, Masafumi; Miyazaki, Motoichi

doi: 10.1002/bmc.1130070505pmid: 8305856

A high performance liquid chromatographic (HPLC) method with fluorescence detection after on‐line reduction for the determination of 1‐nitropyrene (NP), 1‐nitrosopyrene (NSP), 1‐aminopyrene (AP) and N‐acetylaminopyrene (AAP) has been developed. The reduction efficiency of NP and NSP on a zinc column was found to be higher than that of an electrochemical reducer. Using a HPLC equipped with a zinc column (4.0 mm i.d. × 10 mm) and an imidazole/HCIO4 (pH 6.8):acetonitrile mobile phase, detection limits (S/N = 3) of 20‐30 fmol for NP, NSP and AP and 350 fmol for AAP were obtained. NP, NSP and AP were determined in the incubation mixture of NP and Salmonella typhimurium, YG1021, by this method. Time course studies showed that a large ratio of NP was metabolized in the pre‐incubation step of the Ames test.

Schell, Debra A.; Bode, Ann M.

doi: 10.1002/bmc.1130070506pmid: 8305857

Reliable measurement of the reduced and oxidized forms of ascorbic acid (AA) is challenging because they are highly reactive and unstable compounds. Detection of small amounts of AA and dehydroascorbic acid (DHAA) is essential for determining the biochemical function of the vitamin. While a variety of techniques exist for measurement of AA with detection limits in the millimolar range, a need exists for highly reliable assessment of picomolar levels of AA and DHAA in tissues. The present study presents a method for measuring AA and DHAA that combines high performance liquid chromatography with the advantages and increased detection limits and selectivity available with coulometric electrochemical detection. The difference between AA and „total AA”︁ in tissue samples gives an assessment of DHAA concentration. Verification of the reliability of assay is by the successful linear recovery of exogenously added AA and DHAA in tissue homogenate. Optimal conditions for reducing DHAA in tissue samples include a pH of 7.2, reaction time of 10 min, reaction temperature equal to room temperature, and a 10 mM concentration of the reducing agent, β‐mercaptoethanol. AA and DHAA are measured in several mammalian tissues using the method presented.

Alemany, G.; Gamundí, A.; Nicolau, M. C.; Saro, D.

doi: 10.1002/bmc.1130070507pmid: 8305858

A sensitive (up to nanogram level) method for resolving a cannabinoid mixture in plasma is described. Cannabinoids were extracted with a C‐18 Sep Pak cartridge and derivatized with dansylchloride. Then the derivatives were resolved on thin layer HPTLC silica plates which were developed and quantified by fluorescence densitometry at 340 nm.

Imai, Kazuhiro; Fukushima, Takeshi

doi: 10.1002/bmc.1130070508pmid: 8305859

L‐ and D‐Amino acids (Leu or Phe) were derivatized with fluorogenic reagents, 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F), 4‐(N,N‐dimethylaminosulfonyl)‐7‐fluoro‐2,1,3‐benzoxadiazole (DBD‐F), 4‐aminosulfonyl‐7‐fluoro‐2,1,3‐benzoxadiazole (ABD‐F) and 5‐(N,N‐dimethylamino)naphthalene‐1‐sulfonylchloride (DNS‐Cl), and separated on a Pirkle type column, Sumichiral OA 2500 (S) ((S)‐1‐naphthylglycyl‐3,5‐dinitrophenylamide silica gel) with a mobile phase of 20 mM ammonium acetate in methanol. The fluorometric detection of the derivatives was made at 530 nm, 590 nm and 530 nm with excitation at 470 nm, 450 nm, 450 nm and 350 nm, respectively. The former three derivatives of the enantiomers were separated well from each other; The as for each NBD‐, DBD‐ and ABD‐derivative of L‐ and D‐Leu were 1.10, 1.11 and 1.10, respectively. However, the DNS derivatives of L‐and D‐Leu were not separated (separation factor, α = 1.0). All NBD‐ and ABD‐derivatives of L‐ and D‐Phe were also well separated (αs were 1.18, 1.17 and 1.16, respectively), while DNS‐L‐ and ‐D‐Phe were barely separated (α = 1.04). These data suggest that the 2,1,3‐benzoxadiazole (benzofurazan) moiety is very effective and preferable to the dimethylaminonaphthalene sulfonyl (DNS) structure for the separation of enantiomers of amino acids derivatized with benzofurazan reagents. No recemization of each enantiomer occurred during the derivatization reaction with NBD‐F, DBD‐F and ABD‐F at pH 8.0 and 60°C for 2 min, at pH 9.3 and 60°C for 30 min and pH 9.3 and 60°C for 60 min, respectively, meaning that all benzofurazan type fluorogenic reagents are useful for sensitive determination of amino acid enantiomers.