The Method

3. Polyethylene glycols as marker substances for the labelling of addicts' urine

Tampering with samples is a serious problem in connection with the detection of drugs in urine. One of the most common attempts in this regard is the exchange of the drug-contaminated urine with clean urine from another person. For this reason, urine samples must normally be taken under supervision of the doctor, which can lead to various problems in clinics, not least to a deterioration of trust in the relationship between doctor and patient. A marker substance that is taken orally is quickly excreted through the kidneys and does not occur in natural urine offers a solution for these problems.

Principle of enteral marking in urine for drug testing

A substance that is harmless for the patient and easily detectible in bodily fluids is taken orally.
The marker substance is absorbed into the blood in the intestinal lumen and quickly excreted through the kidneys.
The substance analysed together with the relevant drug in the laboratory.

Requirements for a clinical use of marker substances

• No need for time-consuming preparation of the sample or the patient.
• No prolonged waiting periods.
• The amount of substance to be taken for marking should be as small as possible.

• The marker substance should not have any distinct taste.

Sugars have been found unsuitable as marker substances, as they might occur in natural urine, require some preparation of the patient and tend to have long waiting periods (Fordtran et. al. 1967). A better alternative was found in polyethylene glycols (PEG) (Chadwick et. al. 1977). Polyethylene glycols have been used for many years as additives in pharmaceutical products. They are not toxic and show no medical effects whatsoever.

Polyethylene glycols can be determined in urine after a protein precipitation and centrifugation with HPLC. The samples are transferred in an automatic dispenser onto a guard column, where most urine components are separated from the markers that are then transferred onto the actual separating column. Three marker combinations were used in the trials. After separation, chromatograms such as those shown here are obtained. Each signal in the chromatogram is characteristic for a polyethylene glycol of a specific chain length. By combining various chain lengths, it is possible to produce 50 different marker combinations.

HO-CH2-O-CH2-O-CH2-O-CH2-OH n = 4
(n = number of glycol monomers in PEG)
HO-CH2-O-CH2-O-CH2-O-CH2-O-CH2-OH n = 5
HO-CH2-O-CH2-O-CH2-O-CH2-O-CH2-O-CH2-OH n = 6
... n = 7, etc.

Figure 1 shows marker A, while the chromatogram in figure 2 is typical for marker B.

Possible tampering attempts and their detection

Marker solution is spat into urine sample of another person – The marker is given together with sucrose, which is also determined in the lab. Sucrose does not occur in natural urine.
The urine is diluted or mixed with other liquids. – The urine creatinine concentration is determined; the marker concentration normally drops below the limit of detection before the drug reaches this limit.
The urine of the previous patient is taken from the toilet. – The number of marker combinations in the urine is increased.
Acids, alkalines and other chemical substances are added to the sample. – Such manipulations affecting the enzymatic drug analysis are detected by means of CEDIA sample checks.

Procedure of urine sampling with marker.
The patients take the marker in a cup of tea or coffee with sugar.
The urine sample can be taken approx. 30 to 45 minutes later without the need for supervision. During the waiting period, the patient may leave the surgery.

In the lab, the drug content is determined together with the creatinine, a CEDIA sample check is carried out and the glucose concentration after invertase activity is measured in order to determine the sucrose concentration. Urine samples containing glucose are then again examined without adding invertase.

Conclusions:
With the above method, it is possible to determine without doubt whether a urine sample comes from a certain person. The method has been in clinical use for the last five years at the Central Clinical Laboratory of the City of Cologne where it has been applied in about 250.000 tests involving approximately 15.000 patients. No adverse effects could be observed, and there were no non-responders.

Only 3% of the patients must wait as long as 45 minutes until they can give a urine sample. With the patented method, it is virtually impossible to tamper with a sample without being detected, and urine samples can be given without supervision.

Literature:
Chadwick VS, Phillips SF, Hofman AF (1977): I. Gastro-enterol. 73, 241-246.
Fordtran JS, Rector FC, Locklear TW, Ewton MF (1967): J. Clin. Invest. 3, 287-292.
Gauchel G, Huppertz B, Keller R (2003): J. of Chromat. 787, 271-279
Maxton DJ, Bjarnason I, Reynolds AP, Catt SD, Peters DJ, Menzies IS (7986): Clin. Sci. 71, 71-77