immunoassay for the. TDx analyzer. [Abstract]. Clin Chem. 1987;. 33:1570. Abbott. Laboratories. Abbott Diagnostics. Div. Bldg. AP1A-LL. Abbott. Park, IL 60064.
M. Fluorescence polarization hafor the determination of therapeutic drug levels in human plasma. J Anal Toxicol 1981;5:236-40. 7. Schray K, Artz P. Determination of avi6. Jolley munoasaay
din and biotin by fluorescence polarization. Anal Chess 1988;60:853-5. 8. Grenier F, Granados E, Schick B. Enhanced sensitivity immunoassay for the TDx analyzer [Abstract]. Clin Chem 1987; 33:1570. Dinesh Shah’ Vince Salbilla Russell
William Abbott
Broii,
Ill
Laboratories
Abbott Bldg. Abbott
Richerson
Diagnostics
Div.
AP1A-LL
Park,
‘Author
Improved
IL 60064
for correspondence.
HPLC
DeterminatIon
Humans
quent
of
Urinary Hydroxylysine Glycosides to Study Turnover Rate of Bone
in
and Rats
Galactosyl-hydroxylysine
(GHYL)
products of posttranscriptional monoand di-glycosylations of the hydroxylysine (HYL) residues of procol]agen a-chains. The reactions are catalyzed by two specific enzymes, hydroxylysyl-galactosyltransferaseandgalactosylhydroxylysylglucosyltransferase (1), the former linking
the latter
elongating
procedure-that
measurement
sides
in rat chromatographic
urines
shortening
of the
(Fig.
and glucosyl-galactosyl-hydroxylysine (GGHYL) are the respective
and
extraction
low the
To the Editor:
galactose
method for determining these glycosides in human urine (8) to study the effectiveness of the GGHYL/GHYL ratio in evaluating human bone turnover. The ovariectomized rat represents a generally acknowledged animal model for studying postmenopausal bone loss (9). Therefore, we wanted to use our HPLC method to evaluate the possibility of using the GGHYL/GHYL ratio as a marker for monitoring rat bone turnover. However, we encountered the following problems: (a) difficulties in standardizing the pH of the diluted urinary specimens because of their extreme variation in ionic strength, and (b) unsatisfactory chromatographic separations because of several peaks of interfering unknown compounds. Here we describe some modifications of our previous method-mainly specimen treatment and the subse-
1). Specifically,
analysis we
use
and,
consequently,
quantitative We also
and GHYL; this qualitative improvement allowed us to modify the original solvent program, shortening the runtime per assay from 60 to 42 mm (Fig.
1). The modified HPLC procedure is as follows: To analyze HYL glycosides by reversed-phase chromatography, we use precolumn derivatization with 4dimethylaminoazobenzene-sulfonyl chloride (DABS-Cl), a C18 15 x 0.46cm (i.d.) ODS2 Spherisorb column (3 m particles; Phase Separation, Deesid Clwyd, UK), gradient elution with solvent A (sodium acetate buffer, 50 mmol/L, pH 5.21) and solvent B (acetonitrile), and spectrophotometric detection (436 nm). The flow rate is 0.8 mJ.Imin, and the solvent gradient program takes 25 mm from 20% to 45% B, 1 mm from 45% to 90% B, 5 miii constant at 90% B, 1 mm from 90% to 20% B, and 10 miii at 20% B for column equilibration; the total analysis time is 42 min. Sample treatment involves diluting a 0.5-mL aliquot of rat urine with 9 mL of deionized water and 1 mL of 100 mmol/L acetic acid; 10 mL of the di-
a
run-time now
step
recovery of the analytes. include a washing step with pyridine to remove efficaciously those compounds that interfere in the chromatographic separation with the peaks of interest, GGHYL
glyco-
improved and
loading
improve
al-
of HYL with an separation
the
the
greatest acetic
dilution of the rat urine with acid (see below) that yields a diluted urine sample with a sufficiently low ionic strength (15
confirmation
(4); igfL THCA (the DHHS cutoff for THCA by GC-
MS). Of the 286 specimens that screened positive at 100 g/L, all were confirmed positive by GC-MS (>15 p.gIL THCA). For the 115 specimens
Murone
Grazioli1
H.S.
that screened negative at
positive at 50 &gIL but the 100 ig/L cutoff, 97 were confirmed positive by GC-MS (>15 pgfL THCA), 16 were not confirmed, and 2 contained THCA at 60
28
