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Journal of Analytical Toxicology 2013;37:652 –658
doi:10.1093/jat/bkt086
Article
Disaccharides in Urine Samples as Markers of Intravenous Abuse of Methadone
and Buprenorphine
Hilke Jungen, Hilke Andresen-Streichert, Alexander Müller and Stefanie Iwersen-Bergmann*
Department of Legal Medicine, Toxicology, University Medical Center, Butenfeld 34, Hamburg 22529, Germany
*Author to whom correspondence should be addressed. Email: [email protected]
Methadone and buprenorphine are commonly used as oral substitutes in opiate maintenance programs to treat persons who are dependent on heroin. During these programs, patients are not allowed
to continue using illicit drugs. Abstinence can easily be monitored by
urine tests with immunochemical methods. It is well known that the
intravenous abuse of heroin substitutes like methadone or buprenorphine has become common as well. The methadone-prescribing physician has no opportunity to check whether the opiate maintenance
treatment patient takes his substitution medicines orally as intended or
continues with his intravenous misuse now substituting the methadone instead of injecting heroin. In Germany, substitutes are available
as liquids and tablets that contain carbohydrates as adjuvants.
Sucrose is used to increase viscosity in liquids, while lactose is
needed for pressing tablets (e.g., Methaddictw and Subutexw). In case
of oral ingestion, disaccharides are broken down into monosaccharides by disaccharidases in the small intestine. These monosaccharides
are absorbed into the blood stream by special monosaccharide transporters. Disaccharidases do not exist in blood, thus sucrose and
lactose are not split if substitute medicines are injected intravenously.
Our assumption, therefore, was that they are excreted unchanged in
urine. We investigated a method for the detection of disaccharides in
urine as markers of intravenous abuse of substitutes. Urine samples of
26 intravenous substitute abusers showed all positive results for
lactose (76.9%) and/or sucrose (73.1%). The method is assumed to be
useful to detect intravenous abuse of substitutes.
Introduction
Opiate maintenance treatment (OMT) for heroin-addicted
persons started in the 1990s in Germany and the number of
treated persons has increased continuously. In 2011, 76,200
patients took part in German OMT programs. The total number
of opioid users receiving substitution treatment in Europe is estimated to be 730,000. Methadone is the most commonly prescribed medication given to up to three quarters of the clients,
while buprenorphine is prescribed to most of the remaining
clients (1). Substitution with either methadone or buprenorphine provides heroin-addicted patients with an opportunity to
recover from opiate addiction. In Germany, the patients are
medicated with racemic methadone, levomethadone or buprenorphine. Methadone is prescribed in most of the cases (54.8%),
followed by levomethadone (25.4%) and buprenorphine (19.2 %)
(2). Substitutes have to be taken orally each day under the surveillance of a physician or a pharmacist to avoid the occurrence of
withdrawal symptoms. In 1998, the German Narcotics Act was
changed to allow the specially licensed methadone-prescribing
physician to prescribe so-called ‘take-home doses’ for up to 7 days
under certain conditions. These ‘take-home doses’ can be taken
by the patient without supervision. The intent of this policy is to
support and strengthen the rehabilitation of a responsible patient
in the course of treatment. Using this strategy, it is hoped that the
patient will learn to take control of his addiction and will take the
medication only as a way of preventing withdrawal symptoms.
With the increase in the number of patients who receive substitutes and the introduction of ‘take-home doses’, the availability
of methadone on the black market grew (3). Intravenous abuse of
methadone is not uncommon; we know this from fatal cases
where needles with methadone solution were found beside the
drug addict (4), other reports from drug-related deaths (5, 6) or
reports from the drug scene in Germany (7) Australia (8, 9) and
Switzerland (10).
Substitutes are abused by non-substituted individuals, but also
by persons who take part in OMT (3, 7). Reasons for this substitute
consumption as given by the questioned consumers include the
lower price of methadone compared with heroin, lack of availability of heroin or inadequate dosage of prescribed opioids (7, 11).
Until now, patients taking part in OMT with ‘take-home doses’
could not be monitored for the way in which way they administered their substitutes; but information about the route of administration is useful for the attending physicians because it shows
whether patients are compliant to the therapy concept. The aim of
this study was to assess the actual route of methadone or buprenorphine consumption. This was attempted by the determination of
disaccharides as ‘marker substances’ in the urine of drug addicts.
In Germany, substitutes are available as liquids and tablets. In
both medicines, carbohydrates are contained as adjuvant. Table 1
provides an overview of the ingredients of the most common tablets
and formulations prescribed as substitutes in Germany. Sucrose is
used to increase viscosity in liquid methadone preparations (e.g.,
L-Polamidonw together with Viskose Grundlösung NRF (contains
22 g sucrose in 100 mL) (12) or with Sirupus Simplex (contains
64 g sucrose in 100 mL), while lactose is needed for pressing tablets
(e.g., Methaddictw and Subutexw). In case of oral ingestion, both disaccharides are catabolized by carbolytic enzymes in the small intestine. Disaccharidases split sucrose into glucose and fructose and
lactose into glucose and galactose (Figure 1). These monosaccharides are absorbed into the blood by special monosaccharide transporters (13–15). In blood, disaccharidases do not exist. Therefore
sucrose and lactose cannot be split apart if substitutes containing
disaccharides are injected intravenously. They are assumed to be
excreted unchanged in urine. Disaccharides in urine may be used as
‘marker substances’ for intravenously injected disaccharide containing formulations of methadone and buprenorphine.
Materials and method
Reagents and materials
Glucose (Ph. Eur. 6.0), lactose monohydrate (Ph.Eur.6.5) and sucrose
(Ph. Eur. 6.3) were purchased from Caelo (Hilden, Germany),
# The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Table I.
Ingredients of the most common tablets and formulations prescribed as substitutes in Germany.
Drug/formulation
w
L-Polamidon
L-Polamidonw þ Sirupus Simplex or Racemic
Methadone þ Sirupus Simplex
L-Polamidonw þ Viskose Grundlösung NRFor
Racemic Methadone þ Viskose Grundlösung NRF
Methaddictw
w
Subutex
Suboxonew
Active ingredient
Levomethadone
Levomethadone
Methadone (R/S)
Levomethadone
Methadone (R/S)
Methadone (R/S)
Buprenorphine
Buprenorphine
Naloxone
Adjuvant
Carbohydrates
Other
None
Sucrose
Parabenes, betaine hydrochloride, glycerol 85%, water
Parabenes, betaine hydrochloride, glycerol 85%, water
Sucrose
Hydroxyethylcellulose 400, glycerol 85%, citric acid, potassium sorbate, water
Lactose (large amounts)
Sucrose (small amounts)
Lactose
Lactose (168 mg per tablet)
Cellulose, magnesium stearate, maize starch
Mannitol, maize starch, Povidon K30, citric acid, sodium citrate, magnesium stearate
Mannitol, maize starch, Povidon K30, citric acid, sodium citrate, magnesium stearate,
potassium acesulfame
Figure 1. Breakdown of lactose and sucrose by disaccharidases in the brush border membrane in the small intestine into the monosaccharides galactose, glucose, and fructose
after oral intake (a). Presumed elimination of unchanged disaccharides after intravenous abuse (b).
Disaccharides as Markers for Intravenous Abuse 653
D-galactose
was purchased from C. Roth (Karlsruhe, Germany) and
was purchased from Becton, Dickinson and Company
(Sparks, USA). Benzoyl chloride was obtained from Sigma-Aldrich
(Seelze, Germany), while trehalose, tert-butyl-methyl ether (for analysis), water (LiChrosolvw for chromatography) and n-pentane p.a.
were obtained from Merck (Darmstadt, Germany). Acetonitrile
(ultragradient HPLC grade), methanol p.a. and sodium hydroxide p.a.
were purchased from J.T. Baker (Griesheim, Germany).
D-xylose
Standards
Glucose, sucrose, trehalose, fructose and galactose standard
solutions (1 mg/mL) were prepared by dissolving 10.0 mg of the
respective sugar in 10 mL of distilled water. Xylose standard solution (10 mg/L) was prepared by dissolving 100.0 mg in 10 mL
of distilled water. Lactose monohydrate (1 mg/mL) was prepared by dissolving 10.53 mg in 10 mL of distilled water. The
standards were stable for at least 6 weeks when stored at 48C.
Specimen collection
The anonymous sample group consisted of 26 subjects aged
between 26 and 53 years who self-administered methadone,
buprenorphine or heroin intravenously in a drug consumption
room called ‘Drob Inn’, Hamburg, Germany. Users who visited
the Drob Inn and intended to consume their drugs intravenously
were asked by the staff whether they would be willing to deliver
a urine sample for research. Urine samples were obtained 15 –
30 min after intravenous administration of the respective drugs.
Samples were stored as soon as possible at 48C until they were
delivered to our laboratory and stored at 2208C until analysis.
Control group
The control group consisted of 30 healthy subjects (laboratory
staff and students) aged between 22 and 54 years who ingested
20 g lactose and 20 g sucrose orally in one dose. Persons
included in the control group were known to not be illicit drug
consumers. Urine specimens were collected about 30 –40 min
after the ingestion of the sugars and additionally about 2– 3 h
later. All the specimens were stored at 2208C until analysis.
Immunoassays
Urine samples of the intravenous consumer group and the control
group were analyzed for the presence of methadone, buprenorphine, opiates, cocaine and benzodiazepines using standard immunoassay screening tests (CEDIA DAU, Thermo Fisher Scientific,
Middletown, USA) on a Hitachi 912 automatic analyzer. The
samples were also tested for pH and creatinine (Jaffe method).
Sample preparation
After the addition of 20 mL xylose (200 mg/L) and 20 mL trehalose (20 mg/L) as internal standards, 1 mL of urine was precipitated with 2 mL of acetone, vortexed and centrifuged (10 min,
3000 U/min). Supernatant was evaporated to dryness by vacuum
centrifugation for 90 min at 508C and 2 mbar. Derivatization was
performed by ultrasonication for 1 h at 608C after addition of
0.5 mL of 5 M sodium hydroxide and 50 mL of benzoyl chloride
to the residue. A liquid –liquid extraction of derivatized
654 Jungen et al.
carbohydrates was performed with 2 mL of n-pentane. The
n-pentane phase was evaporated to dryness under a stream of nitrogen (at 408C) and reconstituted in 0.1 mL of methanol. An
aliquot of 30 mL was injected into the HPLC system.
HPLC analysis
Analysis of the extracts was performed on a Thermo HPLC-DAD
system using a Thermo UV 6000 detector, Spectra Phoresis SN
4000 interface, a Thermo Spectra System P 4000 pump and a Merck
Hitachi AS 2000 autosampler, software Chromquest 4.2.32 version
3.1.6. The benzoylated carbohydrates were separated on a Varian
Polaris 5 C18 250 4.0 mm column at a flow rate of 1.0 mL/min
and a detection wavelength 230 nm. The mobile phase was composed of acetonitrile, water and tert-butyl-methyl ether (60:24:8).
Validation
The HPLC-DAD method was fully validated according to the
GTFCh Guidelines (16), using the Valistat 2.0 statistics program
(17). In order to evaluate method selectivity, blank urine samples
from six different sources were prepared as described, but without
adding any analyte or internal standards. Furthermore, blank
samples containing only the internal standards were extracted and
analyzed. When analyzing urine samples spiked with opioids
(methadone and buprenorphine), benzodiazepines (diazepam, flunitrazepam and clonazepam) and opiates (morphine and codeine,
6-monoacetylmorphine), no interferences were found.
Calibration was performed by linear regression analysis of arearatio (disaccharides/trehalose) to the calibrator concentration. A
7-point calibration curve was prepared using 1–100 mg/L concentrations of sucrose and lactose in blank urine (calibrators: 1,
5, 10, 20, 50, 70, 100 mg/L). Three quality control samples (8, 40
and 80 mg/L) were analyzed in duplicate over eight days. These
samples were used to determine accuracy, precision and reproducibility. The spiked urine samples were stored for 1, 3, 7 and
10 days at 2208C, 48C or 208C to assess storage stability.
Furthermore blank urine samples with 40 mg/L galactose, fructose and glucose were prepared and analyzed to obtain knowledge about possible interferences by monosaccharides. It was
thought that the possibility might exist that monosaccharides
could play a role in case of decomposition of the disaccharides.
Results
Validation results
The method for analysis of disaccharides in urine was developed
and validated. This was quite difficult because of the need to
isolate hydrophilic analytes out of a hydrophilic matrix. By doing
the derivatization step first and then following it with the extraction, the analysis was finally successful. The benzoylated carbohydrates sucrose, lactose, glucose and galactose were extracted,
separated and detected by HPLC-DAD. Figure 2 shows a typical
chromatogram with detected disaccharides and monosaccharides.
The calibration curves fitted to a linear regression function
with r 2 . 0.99; inter- and intra-day imprecision was less than
+15%. Further validation results are shown in Table 2. Storage
experiments showed the disaccharides to be stable in urine at
2208C. No monosaccharides were detectable even after a
Figure 2. HPLC/DAD chromatogram. Retention times of the benzoyl-derivatives are 17.59 min for lactose, 21.54 min for sucrose, 7.23 min for the first internal standard xylose,
20.23 min for the second internal standard trehalose, 8.58 min for glucose and 10.61 min for galactose.
storage period of 10 days. At 48C the disaccharides showed a
decline of about 17% (sucrose) and 43% (lactose) within 10
days. At 208C sucrose was reduced by 24% and lactose even by
57% (Figure 3). After storage at 48C and 208C, monosaccharides
were detectable in the disaccharide-spiked samples.
Analysis of urine samples of intravenous drug abusers
and control group
After successful method validation, the urine samples of the
control group were analyzed. No disaccharides, no monosaccharides and no drugs of abuse were detected in the urine samples of
the persons of the control group who ingested disaccharides
orally. The method was then applied to the quantitative analysis of
sucrose and lactose in urine samples of known drug addicts who
consumed drugs of abuse or substitutes intravenously. We obtained these samples from a drug consumption room, called Drob
Inn. It is an institution where drug addicts may take their drugs
under some kind of supervision combined with the offer of
medical or psychosocial support. Drug consumption rooms aim
to reduce the presence of high-risk drug use in the public eye.
Immunological analysis of the urine samples of the intravenous
substitute abusers showed positive results for methadone
(53.8%), opiates (53.8%), benzodiazepines (50%), cocaine (69%)
and buprenorphine (38.5%). No sample was negative. The majority of samples (76.9%) were positive for two or more parameters.
Table 3 gives the detailed results. In all samples, creatinine and
pH values were found to be in a physiological range.
All the urine samples were tested positive for lactose or
sucrose. In 23.1% of specimens only sucrose was detected, while
lactose alone was found in 26.9% of the analyzed samples. Both
disaccharides were positive in 50% of the cases. Detailed quantitative results for the disaccharides in combination with immunochemical results are given in Table 4. Both disaccharides were
found in a concentration range from 1.5 mg/L to .100 mg/L.
Most of the samples showed results .50 mg/L.
Disaccharides as Markers for Intravenous Abuse 655
Table II.
Evaluation data of sucrose and lactose in urine including intra-day precision (CV%, n ¼ 6), inter-day precision (CW%, n ¼ 6), accuracy (%, n ¼ 6) and recovery
Lactose
Limit of detection [mg/L]
Limit of quantification [mg/L]
Nominal concentration [mg/L]
Characteristics
Mean
SD
Coefficient of variance [%]
Accuracy
Variance
Bias [%]
Intra-day precision
SD
RSD [%]
Inter-day precision
SD
RSD [%]
Extraction efficiency [%]
Processed sample stability [%]
Sucrose
QC1 8.0
0.3
0.6
QC2 40.0
QC1 8.0
0.3
0.5
QC2 40.0
QC3 80.0
QC3 80.0
7.93
0.75
9.5
39.46
4.65
11.79
79.19
8.34
10.53
8.48
0.87
10.2
40.18
4.68
11.66
80.71
9.48
11.75
20.07
20.9
20.54
21.4
20.81
21.0
0.48
6.0
0.18
0.5
0.7
0.9
0.51
6.5
3.78
9.6
7.46
9.4
0.96
11.4
3.05
7.6
10.08
12.49
0.76
9.6
62.8
9.3
4.65
11.8
62.2
2.8
8.1
10.62
0.96
11.4
60.5
2.7
4.78
11.9
65.6
3.1
10.08
12.49
QC—quality control; CV—coefficient of variance.
Figure 3. Decrease in sucrose and lactose concentration in urine specimens stored for 10 days. Samples with a concentration of 100 mg/L sucrose and lactose were stored at
2208C, 48C and 208C. The disaccharide concentrations were determined in duplicate; means are displayed.
Discussion
The results show the developed method to be suitable for the detection of carbohydrates in urine. Sucrose and lactose can be
determined simultaneously. The monosaccharides, galactose and
glucose, can chromatographically be separated and detected, but
it was not possible to detect fructose in an adequate manner.
Therefore, a complete validation for monosaccharides was not
performed, only qualitative analysis was done. Storage experiments had shown the disaccharides to be stable at 2208C for 10
days. In these samples no monosaccharides were detectable even
after storage of 10 days. After storage at 48C and 208C, monosaccharides were detectable in the samples. The detectability of
monosaccharides in all samples is, therefore, to be assumed by
the degradation of disaccharides. Actually, there was no urine
656 Jungen et al.
Table III.
Positive immunochemical results of urine samples
Positive immunochemical results n ¼ 26
Methadone
Methadone alone
þ Benzodiazepines
þ Opiates
þ Buprenorphine þ benzodiazepines
þ Benzodiazepines þ opiates
Buprenorphine
Buprenorphine alone
þ Benzodiazepines
þ Opiates
þ Methadone þ benzodiazepines
þ Benzodiazepines þopiates
Opiates
Opiates alone
þ Benzodiazepines
þ Other combinations see above
14
0
4
3
1
6
10
4
3
1
1
1
14
2
1
11
Table IV.
Immunochemical results and results for disaccharides in urine samples; cutoff concentration: buprenorphine 5 ng/mL, methadone 200 ng/mL, opiates 200 ng/mL and benzodiazepines 200 ng/mL
Case
Sex
Age
Immunoassays [ng/mL]
Buprenorphine
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
f
f
m
m
f
m
m
m
f
m
f
f
f
f
m
n.a.
n.a.
n.a.
m
m
m
f
m
f
n.a.
n.a.
26
26
41
32
30
35
50
43
43
25
23
35
23
35
37
n.a.
n.a.
n.a.
35
27
53
35
29
33
n.a.
n.a.
Disaccharides [mg/L]
Methadone
322
487
561
Benzodiazepines
1301
710
.5,000
Opiates
Lactose
.2,000
.2,000
.2,000
.2,000
.2,000
.2,000
13
25
.200
.600
37
.200
.200
.200
358
375
.600
4,835
.5,000
.5,000
1,324
.5,000
.2,000
.2,000
.5,000
87
.200
.200
8
.2,000
1,020
406
411
489
409
366
430
519
.200
94
462
27
572
61
102
132
630
543
350
.5,000
.5,000
.5,000
1,017
920
.5,000
2,397
.2,000
1,592
1,099
.2,000
1,040
13
389
560
133
300
358
47
Sucrose
122
143
61
133
47
62
109
457
311
73
217
326
219
26
358
181
380
212
422
n.a.—not available.
sample from the intravenous consuming drug users containing
no monosaccharides. The sample-taking procedure can explain
these positive results. The samples were collected in the drug
consumer room and were then stored by the staff at 48C as soon
as possible. Until the samples could be stored at 2208C in our
lab, a time span of 8–48 h passed. Unfortunately, it was not possible to deep freeze the urine samples in the consumer room.
All urine samples of the intravenous drug consumer group had
positive findings for disaccharides after the injection of the substitutes, whereas all samples from the control group showed
negative results for disaccharides and monosaccharides after oral
consumption of a higher dose of both disaccharides. Positive
results for carbohydrates are, therefore, not to be anticipated in
healthy persons after oral ingestion. These results suggest that
the method is useful for distinguishing between an intravenous
and an oral use of substitutes. Certainly, in case of intestinal diseases such as ulcerative colitis or Crohn’s disease, this method
might not be applicable, because the mucosal barrier is inadequate and intact disaccharides can be absorbed from the small
intestine (13, 14).
The detection of sucrose alone (42.9% of methadone positive
specimens) suggests the intravenous abuse of liquid methadone
formulations which contain only sucrose (and no lactose) as a
viscosity enhancer. We checked the declarations of many tablets
as given by the manufacturer and all of them contained only
lactose. The only exception was Methaddictw. Methaddictw
tablets contain lactose and a small amount of sucrose. The detection of big amounts of lactose and small amounts of sucrose in
urine, therefore, strongly suggests the intravenous application of
Methaddictw tablets. Some consumers prefer tablets due to their
known fixed methadone content per tablet (7). It has to be kept
in mind that the sample group did not consist of substituted
patients. Actually, we do not know where the consumers
obtained their methadone and buprenorphine, but it is astonishing that 23 out of 26 intravenous drug abusers consumed substitutes and only 14 persons opiates. As was shown by the
immunochemical results, the sample group consists of polyvalent multi-drug abusers. Not only methadone, but opiates, benzodiazepines and cocaine were found. Other drugs of abuse were
not tested. Therefore, the results for disaccharides as markers
for intravenous methadone abuse can be influenced by additional intravenous intake of opiates or benzodiazepines. One sample
showed only opiates and benzodiazepines to be positive and
lactose alone. Street heroin is often blended with lactose and
benzodiazepine tablets contain lactose as well. Substituted
patients are not allowed to consume opiates or benzodiazepines
intravenously in addition to their methadone therapy. Therefore,
this should present no problem when substituted patients
without parallel consumption of other drugs are tested for disaccharides during a compliance control.
The results of this study reveal that intravenous use of substitutes like methadone and buprenorphine takes place and has
to be addressed by the attending doctors and therapists.
Intravenous substitute users blog in internet platforms that it is
unproblematic to inject methadone tablets dissolved in water or
liquids, even those with viscosity enhancers, directly. Some
doctors might not be aware of the fact that patients do indeed
inject those partially colored viscous liquids.
The established method might also be useful for assessing
drug-related deaths. An intravenous application of methadone or
buprenorphine results in a higher toxicity due to the fast initial
accumulation in brain opiate receptors. Further investigation is
Disaccharides as Markers for Intravenous Abuse 657
necessary to adapt the method for a detection of carbohydrates
in postmortem urine and blood samples.
A more detailed study is in progress to assess the length of
time disaccharides are detectable in urine after consumption.
Most of the samples showed high concentrations (.50 mg/L
sucrose and/or lactose). It is, therefore, anticipated that detection can be achieved for several hours after consumption.
Conclusion
Substitution medicines like methadone and buprenorphine are
abused intravenously in the drug scene. Disaccharides were
shown to be appropriate specific markers to identify an intravenous uptake of disaccharides via lactose-containing buprenorphine
or methadone tablets or sucrose-containing liquids with methadone. The method is a suitable complement to routine screening
for drugs of abuse if methadone-prescribing physicians suspect
that their patients are abusing the substitutes intravenously.
Furthermore, such determinations might be helpful in assessing
drug-related deaths because the intravenous application is associated with a higher risk of fatal respiratory depression.
Acknowledgments
We thank Peter Möller and his team from Drob Inn - Jugendhilfe
e. V. in Hamburg for their help in getting urine samples from
drug addicts.
Dedicated to Professor Dr. Achim Schmoldt, Hamburg,
Germany, on the occasion of his 75th birthday.
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