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The detection of disaccharides in urine is under investigation to act as a marker for intravenous abuse of disaccharide formulations, like liquid methadone with syrup sucrosemethadone tablets lactose and sucroseor buprenorphine tablets lactose. As the detection time in urine has not yet been investigated and a routine method for detecting disaccharides is still lacking, a study was performed to estimate the window of detection in urine after intravenous consumption of disaccharides. The method was applied to urine samples of intravenous substitute consumers, with urine being sampled before intravenous use of substitutes and approximately 30 minutes later.
Twenty users provided information regarding their most recent prior intravenous consumption. Disaccharides were detectable in all 20 urine samples about 30 minutes after consumption. The study showed that the validated LC-MSMS method with an easy and fast workup is usable for daily routine in the laboratory. It might be helpful for methadone and buprenorphine prescribing physicians to check whether the opiate maintenance treatment patient takes his or her substitution medicines orally as intended, or continues with intravenous misuse by injecting substitution medicines instead of heroin.
The most common form of treatment of heroin dependence is opiate maintenance treatment OMT. The baseline goal of OMT is to reduce mortality, especially by reducing infectious diseases and serious intoxication 1. Principle of the treatment is to replace intravenous heroin use by controlled oral intake controlled opiate dependence in order to avoid the experience of withdrawal symptoms.
The illicit use of other addictive substances, especially heroin, puts patients at risk and must therefore be monitored by urine testing. Beside opiates, cocaine, amphetamines, benzodiazepines and cannabinoids are further parameters to be tested for routinely. Misuse of the substitutes methadone or buprenorphine has been reported and is not uncommon.
Substitutes are abused by non-substituted individuals, but also by persons who take part in OMT. Besides oral intake, methadone and buprenorphine are consumed intravenously like heroin by drug abusers 2 — Until now the methadone-prescribing physician has no opportunity to check whether the OMT patient takes his or her substitution medicines orally as intended, or continues with intravenous misuse by injecting methadone instead of heroin.
We could show in a study that disaccharides like lactose and sucrose were detectable in urine of 26 persons after intravenous application of disaccharide-containing methadone formulations, such as methadone with sirupus simplex sucrose or methadone tablets lactose and sucrose.
The oral intake of disaccharides did not produce positive in urine 8. The explanation is that in case of oral ingestion, disaccharides are broken down into monosaccharides by disaccharidases in the small intestine, which are absorbed into the blood stream by special monosaccharide transporters. Disaccharidases do not exist in blood, thus sucrose and lactose are not splitted if substitute medicines are injected intravenously. Our assumption, therefore, was that they are excreted unchanged in urine. On the basis of these preliminarythe detection of disaccharides in urine had been suggested by our group as a promising marker of intravenous use of methadone formulations containing disaccharides.
Our method was suitable for the detection of disaccharides and monosaccharides glucose, galactose in urine samples, which was especially important How to inject liquid methadone the stability study of disaccharides. However, the method was too time-consuming for routine laboratory use to detect disaccharides in urine.
The preliminary highlighted the need to develop an easy method for disaccharide detection in urine. Furthermore, we got the chance to get further urine samples of intravenous users of methadone and buprenorphine.
For the current study, we chose a new modus of urine collection. The users were asked what kind of substitution medicine they intended to consume and when they consumed intravenously for the last time. As a complement to the questionnaire, two urine samples were collected from each volunteer before and short time after intravenous drug consumption at two separate times. The aim of this measure was to assess the time window after intravenous use inside which the detection of disaccharides can be assumed.
But in a retrospective study of methadone-related death concerning intravenous abuse of methadone, we recognized some cases to show very low amounts of disaccharides in urine This might be due to post mortem degradation of disaccharides. Another explanation might be high dosed oral intake of disaccharides. All solvents and reagents for sample preparation were of specified ACS grade.
Water, acetonitrile, ammonium acetate and formic acid were purchased from Fluka Munich, Germany. Lactose monohydrate Ph. Blank urine was obtained from the laboratory staff on a voluntarily basis. The blank urine samples were analyzed by immunoassay screening tests for the presence of drugs of abuse. The fold and fold diluted standard solutions concentration 0. For the internal standard, 1 mg of d2-sucrose was weighed accurately into a mL volumetric flask and made up to 10 mL with water. An aliquot of 0.
After vigorous mixing, acetone 1 mL was added. The extracts were reconstituted in 0. The flow rate of the mobile phase was 0. With approval from the Regional Ethics Committee, urine samples from an anonymous donor group collected in a drug consumption room were examined for disaccharides and abused drugs.
Users who visited Drob Inn and intended to consume their drugs intravenously were asked by the staff if they would be willing to anonymously deliver two urine samples for research. If they agreed, a very short questionnaire was to be filled out providing information about: the kind of drugs they intended to consume at the Drob Inn, the time at which they last consumed drugs, and which kind of drugs they had last consumed. Urine samples were obtained anonymously 30—60 min before and after intravenous administration of the respective drugs.
The samples were also tested for pH and creatinine Jaffe method. Two buprenorphine positive samples 9 and 20 with additional positive for sucrose which could not be explained were analyzed by our own in-lab LC—MS-MS method for drugs of abuse which includes methadone and EDDP in urine samples in order to exclude false negative immunochemical. In brief, 1 mL urine was spiked with internal standards corresponding deuterium labeled compoundsbuffer and beta glucuronidase enzymes.
In MRM mode, one transition for each analyte was detected. In order to evaluate method selectivity, blank urine samples from six different sources were prepared and analyzed as described, but without adding any analyte or internal standards. Furthermore, blank samples containing only the internal standards were extracted and analyzed. Urine samples were spiked with opioids methadone, EDDP, buprenorphine, norbuprenorphinebenzodiazepines diazepam, nordiazepam, oxazepam, flunitrazepam, 7-aminoflunitrazepam, clonazepam and opiates morphine, codeine, 6-monoacetylmorphine and analyzed for interferences with the method.
Samples spiked with internal standard and analytes after extraction were compared with samples spiked with analytes before but internal standard after extraction. All validation samples were prepared from blank urine samples obtained from laboratory staff. Further validation are shown in Table I. Table I. Validation. Quantitative for sucrose are given in Table II. Table II. After successful method validation, the method was applied to the quantitative analysis of sucrose and lactose in urine samples of known drug addicts who consumed substitutes intravenously.
One person reported a mixed consume of methadone tablets How to inject liquid methadone liquid. Immunological analysis of the urine samples, together with the for disaccharides, is given in Table III. In all samples, creatinine and pH values were found to be in a physiological range. Six of the methadone consumers additionally showed positive immunological for opiates, benzodiazepines and cocaine. In these samples, sucrose was found in a concentration range from Last intravenous consumption was stated to have taken place between 1 and 92 h ly.
In How to inject liquid methadone group in 18 out of 20 samples, sucrose was detected from Two samples were completely negative. Detailed quantitative for the disaccharides in combination with immunochemical are given in Table III. Table III. Immunochemical test and quantitative disaccharide in urine. Compared with the first method with HPLC separation of the benzoylated monosaccharides and disaccharides, the simplified workup of urine samples and the detection of underivatized disaccharides with LC—MS-MS is much easier and faster.
Furthermore, the use of isotope sucrose as an internal standard makes the method robust toward matrix effects. The studies on selectivity did not reveal any impact from the frequently considered drugs. In principle, the method should be suitable for analysis during routine control of urine samples of substituted patients.
Sensitivity was not higher with the LC—MS-MS method, but it should be kept in mind that the necessary sample amount was reduced from 1. The quantitative achieved for disaccharides are much higher. The detection of high amounts of sucrose alone suggests the intravenous injection of liquid methadone formulations syrup containing only sucrose and no lactose as a viscosity enhancer. The persons with s 4, 15 and 19 stated that they intended to inject liquid methadone formulations and showed only sucrose positive in their samples.
Person 15 gave the information that he consumed liquid methadone 3 h ago. Unfortunately, it was not asked in the questionnaire if a viscosity enhancer like sucrose syrup was added to liquid methadone. The addition of syrup is recommended but not obligatory in Germany. This might be an explanation for the missing detection of sucrose, although consumption was just 3 h ago. When pure methadone is applicated intravenously, obviously no disaccharides can be awaited in urine.
According to the manufacturer declarations of many tablets methadone, buprenorphine and benzodiazepinesall contain only lactose and no other monosaccharides or disaccharides. All of them showed higher lactose —3, Longer periods of uncooled storage until analysis favor degradation of lactose more than degradation of sucrose and might also influence the proportion of lactose to sucrose 8. Both urine samples of Person 13 showed positive for only lactose Person 20 showed positive for lactose in both samples Person 9 showed positive for lactose in the urine sample directly sampled after intravenous consume Both samples were negative for methadone and EDDP.
The confirmed the negative immunochemical testing for methadone. Therefore, small amounts of sucrose can only be explained by oral intake. Hence, there is a necessity to define a cutoff for both sugars. Last intravenous consumption was stated to be from 1 to 92 h ago. Three persons with a reported last intravenous consume 4, 8 and 12 h ago showed negative. An explanation can be that the statements of the consumers have to be considered as not very reliable.
As told by the staff of the Drob Inn many of them were still intoxicated when they administered their new methadone dose. Therefore, it is not very surprising that not all statements match with the analytical findings. This belongs as well to Person It has to be kept in mind that the reliability of self-reported drug use is limited. Although our preliminary with oral ingestion of 20 g sucrose and 20 g lactose in healthy subjects did not give a hint for relevant renal elimination. It has to be taken into that beside the oral intake of disaccharides by formulations of substitutes additional relevant amounts of sucrose can be ingested by food and beverages.
Therefore, we increased the intake of disaccharides in our current control group. Sucrose concentrations of Immunochemical testing was negative for methadone in these cases. A possible explanation for the detection of sucrose How to inject liquid methadone be a consequence of very high dosed oral intake of sucrose or an increased permeability of the intestinal mucous membrane by non-steroidal anti-inflammatory drugs A reliable cutoff recommendation cannot be given for lactose.
Additional characterization of lactose and sucrose in urine and their time profile in larger populations of healthy subjects, patients with a medication of non-steroidal anti-inflammatory drugs, and patients in OMT are indicated before this test can be considered with a reliable cutoff for clinical use. above the cutoff might be a good decision-making basis for the therapist to do a thorough examination of the substituted patient for fresh needle marks.How to inject liquid methadone
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Patients in methadone maintenance treatment who inject methadone syrup: a preliminary study