Clinical Chemistry 43: 1029-1032, 1997;
(Clinical Chemistry. 1997;43:1029-1032.)
© 1997 American Association for Clinical Chemistry, Inc.
Simultaneous identification and quantitation of codeine, morphine, hydrocodone, and hydromorphone in urine as trimethylsilyl and oxime derivatives by gas chromatography–mass spectrometry
Larry A. Broussard1,a,
Lance C. Presley2,
Thomas Pittman3,
Randy Clouette4 and
Gary H. Wimbish4
1
Department of Medical Technology, LSU Medical Center, New Orleans, LA 70112-2262.
2
LabCorp, Memphis, TN 38118.
3
TF Puckett Laboratory, Hattiesburg, MS 39402.
4
Harrison Laboratories, Midland, TX 79706.
a Author for correspondence. Fax 504-568-6761; e-mail lbrous{at}lsumc.edu
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Abstract
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Following enzymatic hydrolysis of urine, a gas chromatography–mass
spectrometry method for the simultaneous determination of codeine,
morphine, hydrocodone, and hydromorphone uses hydroxylamine to
form oxime derivatives of the keto-opiates (i.e., hydrocodone,
hydromorphone, oxycodone, and oxymorphone). These
trimethylsilyl-derivatized forms no longer interfere with the detection
and quantitation of codeine and morphine. Samples are extracted on
solid-phase columns and quantitated by deuterated internal calibrations
of each analyte with selected ion monitoring. Codeine, morphine,
hydrocodone, and hydromorphone are completely separated, allowing
simultaneous quantitation without interference and a chromatographic
analysis time <9 min.
Key Words: indexing terms: abused drugs • drug monitoring • opiates • forensic toxicology
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Introduction
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Drug testing for opiates under the Mandatory Guidelines for
Federal Workplace Drug Testing Programs requires immunoassay screening
and confirmation by gas chromatography–mass spectrometry (GC/MS) for
morphine and codeine. The immunoassays available for opiate
testing have variable cross-reactivity to codeine, morphine, and other
opiates (1). Detection and quantitation by GC/MS of
opiates such as the keto-opiates hydrocodone, hydromorphone, oxycodone,
and oxymorphone are desirable not only because of their potential
interference with the measurement of codeine and morphine but also
because of their potential for abuse. Several GC/MS methods have been
developed for the analysis of codeine, morphine, or other opiates. The
extraction, derivatization, and detection details of many GC/MS methods
have been reviewed by Goldberger and Cone (2) and Wasels
and Belleville (3). Chen et al. (4) and
Grinstead (5) studied the stability and characteristics of
various derivatives used for opiate analysis.
Problems encountered with some methods include instability of
derivatives, poor chromatography, unsuitable ions and abundances,
incomplete derivatization, derivatization side reactions, inadequate
recovery, loss during hydrolysis, extended run times, and interference
or coelution by other opiates. The potential interference of other
opiates, particularly the keto-opiates hydrocodone, hydromorphone,
oxycodone, and oxymorphone, with the analysis of codeine and morphine
is a major concern. Techniques to improve separation of these opiates
include pretreatment with borohydride (6), sequential
derivatization (7), and multiple ramp temperatures
(8). The method presented here incorporates the use of
hydroxylamine after enzymatic hydrolysis to form oxime derivatives of
the keto-opiates (9)(10), whose derivatized forms do not
interfere with codeine or morphine detection and quantitation. The
procedure includes enzymatic hydrolysis of 2.0-mL urine samples
followed by reaction with hydroxylamine, extraction on solid-phase
columns, and derivatization with
N,O-bis(trimethylsilyl)trifluoroacetamide
(BSTFA).1
Codeine, morphine, hydrocodone, and hydromorphone are separated without
cross-interference and quantitated with deuterated internal standards
and selected ion monitoring (SIM).
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Materials and Methods
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instrument
A Hewlett-Packard (HP) (Palo Alto, CA) 5890 gas chromatograph with
splitless injection and a 5970 mass selective detector were used. The
capillary column used was a J&W Scientific (Folsom, CA) DB-1 [15
m x 0.32 mm (i.d.), 0.25-µm-thick film]. Helium was the
carrier gas at a flow rate of 0.7 mL/min and a linear velocity of 38
cm/s. The temperature program was: initial temperature, 150 °C for
1.5 min; ramp at 20 °C/min to 250 °C; injection temperature,
250 °C; and transfer line, 280 °C.
materials
Codeine, morphine, hydrocodone, and hydromorphone used to prepare
calibrators and controls were obtained from Sigma Chemical Co. (St.
Louis, MO) and Radian (Austin, TX). Deuterated codeine, morphine,
hydrocodone, and hydromorphone (used as internal standards) and
oxycodone, oxymorphone, and norcodeine (used for interference studies)
were obtained from Radian. Hydroxylamine hydrochloride and Helix
pomatia (type H-2) ß-glucuronidase were obtained from Sigma
Chemical Co. BSTFA with 10 mL/L trimethylchlorosilane (TMCS) was from
Regis Chemical Co. (Morton Grove, IL). All other solvents and reagents
were of reagent or HPLC-grade.
extraction and derivatization
A 2.0-mL volume of urine (calibrators, controls, samples) was
combined with 100 µL of a 10 mg/L internal standard solution
(deuterated codeine, morphine, hydrocodone, and hydromorphone) and
100 µL of 2.0 mol/L acetate buffer (pH 4.8) in an appropriately
labeled 16 x 100 mm screw cap tube. All samples were
vortex-mixed. Conjugates were hydrolyzed by the addition of 100 µL of
ß-glucuronidase solution (99.2 U/L) to all calibrators, controls, and
donor samples and incubation at 56 °C for 2 h. After
hydrolysis, extraction derivatization of keto-opiates was performed by
the addition of 100 µL of aqueous hydroxylamine (100 g/L) to each
tube, then heating for 15 min at 56 °C. All tubes were centrifuged,
and the supernatant was placed on solid-phase bonded silica extraction
columns [Varian (Palo Alto, CA) Bond ElutTM or United
Chemical Technology (Bristol, PA) Clean ScreenTM],
previously activated by the sequential addition and elution of 3 mL of
both methanol and deionized water. Columns were then washed by the
sequential addition and elution of 3 mL of water, 2 mL of 0.1 mol/L
acetate buffer (pH 4.0), and 3 mL of methanol and then dried under
maximum vacuum for 5 min. The analytes were eluted by the addition of 3
mL of freshly prepared methylene chloride:isopropyl alcohol:ammonium
hydroxide elution solvent (78:20:2 by vol) at a rate of ~1–2 mL/min.
The eluants were dried under a stream of air at 56 °C. The extracted
residue was reconstituted with 100 µL of BSTFA with TMCS, 10 mL/L,
and the tubes were capped and heated for 20 min at 56 °C. Hexane
(100 µL) was added to each tube and transferred to a correspondingly
labeled autoinjector vial, and 1 µL of this solution was injected in
the GC/MS with a HP 6890 Automatic Liquid Sampler.
data acquisition and analysis
SIM was used, and the data system was a HP DOS
ChemstationTM. The following ions were monitored
(quantitative ions are indicated in parentheses) for the derivatized
analytes: codeine, 234, 343, (371); d3-codeine,
346, (374); morphine, 234, 401, (429); d3-morphine, 417,
(432); hydrocodone, 297, 371, (386); d3-hydrocodone, 300,
(389); hydromorphone, 429, 444, (355); and
d3-hydromorphone, 447, (358). Quantitation was based on a
calibration curve consisting of calibrators (prepared by appropriate
dilutions of a 1 g/L solution of codeine, morphine, hydrocodone, and
hydromorphone in 2.0 mL of drug-free urine) extracted with each batch
of samples. The concentration of the calibrators in the curve were 120,
300, 1000, and 2000 µg/L.
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Results and Discussion
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chromatography
The total ion chromatographs of the trimethylsilyl (TMS)
derivatives of codeine, morphine, and norcodeine accompanied by the
TMS-oxime derivatives of hydrocodone and hydromorphone are shown in
Fig. 1
. The chromatographic peaks are gaussian-shaped and demonstrate
near-baseline resolution. Under these analysis conditions all peaks are
eluted within 9 min. This resolution was maintained on the same column
for >3500 injections. The retention times and relative (to codeine)
retention times of these and other compounds of interest are shown in
Table 1
. A single peak was obtained for each of the keto-opiates in
scan mode, indicating completeness of oxime formation.
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Figure 1. Chromatograph of urine sample containing 1000 µg/L
codeine, morphine, norcodeine, hydrocodone, hydromorphone, and
oxycodone processed through the procedure.
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linearity
The procedure exhibits linearity for all four analytes with an
upper limit of linearity of 2000 µg/L. As part of each analysis, a
best-fit linear regression calibration curve consisting of the four
calibrators and a forced y-intercept of zero is obtained.
Correlation coefficients of 0.995 or greater for the calibration of
each analyte have been obtained on a daily basis for a 9-month period.
precision
Between-run precision (n = 47) around the 300 µg/L cutoff
was determined by daily analysis of samples with target concentrations
of 240 and 360 µg/L. CVs at these concentrations were: 5.4% and
6.0% for codeine, 8.4% and 6.3% for morphine, 4.2% and 6.0% for
hydrocodone, and 4.0% and 6.6% for hydromorphone, respectively.
interference
Possible cross-interference with quantitation of codeine,
morphine, hydrocodone, and hydromorphone was assessed by adding to
urine samples containing each analyte at the cutoff concentration of
300 µg/L another 10 000 µg/L of each potential interferant. For
example, urine samples with codeine and morphine target concentrations
of 300 µg/L were supplemented (in separate samples) with 10 000
µg/L hydrocodone, hydromorphone, oxycodone, oxymorphone, and
norcodeine. In all cases the quantitation of the analyte of interest
was not affected by the presence of the most common potential
interferants (i.e., codeine, morphine, hydrocodone, and hydromorphone
concentrations were within 20% of the target concentration, ion ratios
were within 20% of the calibrators, and there was no chromatographic
interference). These results demonstrate that the presence of large
concentrations (10 000 µg/L) of hydrocodone, hydromorphone,
oxycodone, oxymorphone, and norcodeine does not interfere with the
analysis of codeine and morphine, and the presence of large
concentrations of codeine, morphine, and norcodeine does not interfere
with the analysis of hydrocodone and hydromorphone. In particular
hydromorphone and norcodeine do not interfere with morphine
quantitation, and hydrocodone does not interfere with codeine
quantitation. These interferences are of concern because of common ions
shared when the TMS derivatives alone are used for the analysis of
these opiates.
detection limits (lod/loq)
The limit of detection (LOD) and limit of quantitation (LOQ) were
determined by serial dilution analysis as described below and not by
calculations based on standard deviation ratios. Samples containing
decreasing concentrations of the analytes of interest are assayed in
triplicate. The LOQ is defined as the concentration at which two of the
three specimens meet the criteria that all chromatographic variables
(retention time, peak shape, qualifier ion ratios, etc.) for a positive
sample are acceptable and the concentration obtained is within 20% of
that expected. The LOD is the concentration at which two of the three
specimens meet the criteria required for a positive determination but
the quantitative result does not have to be within 20% of the expected
concentration. The experimentally determined LOD and LOQ for each
analyte were equal according to these definitions. The LODs and LOQs
for codeine, morphine, hydrocodone, and hydromorphone are 50, 100, 75,
and 75 µg/L, respectively. Because LOD and LOQ values may vary
between laboratories, the National Laboratory Certification Program
(Directive 025, May 1993, J.H. Autry) believes that all laboratories
should be able to identify and quantitate drugs at concentrations
between the cutoff and at least 40% of the cutoff. The LOQ and
LOD concentrations obtained here are all below this minimum requirement
(40% of the 300 µg/L cutoff is 120 µg/L).
In conclusion, the above-described method, which allows the
simultaneous quantitation of codeine, morphine, hydrocodone, and
hydromorphone, demonstrates acceptable precision, linearity,
sensitivity, and lack of cross-interference and interference from other
opiates. It has been used in our laboratory for the analysis of >2500
samples in 9 months and has been found to be reliable as demonstrated
by calibrator and control reproducibility and absence of interference.
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Footnotes
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1 Nonstandard abbreviations: SIM, selected ion monitoring; BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; HP, Hewlett-Packard; TMCS, trimethylchlorosilane; TMS, trimethylsilyl; LOD, limit of detection; LOQ, limit of quantitation. 
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References
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Abstract
Introduction
