Confirmatory Testing: Gas Chromatography/Mass Spectrometry

Gas Chromatography/Mass Spectrometry (GC/MS) is a combination of two techniques: Gas Chromatography and Mass Spectrometry. It uses internal standards and provides identification and quantification of drugs and serves as the gold standard in the confirming the abuse of prohibited drugs such as heroin.

PRINCIPLE

Gas Chromatography

Gas Chromatography is a technique used to separate the drugs that may be present in the patient's sample. The chromatography column is a long tubular column where the sample is injected and is swept through the column by means of a gas such as helium. Drugs present are separated from one another since some drugs take longer to pass as compared with others. The chemical characteristics of drugs the time it takes for the drug to travel through the chromatography column. This is referred to as the retention time which is an identifying characteristic of the drug.

Mass Spectrometry

Mass Spectrometry is the detector of Gas Chromatography. When the drug exits from the GC column, it is separated via ionization. The fragments are sorted by mass forming a fragmentation pattern which is also an identifying characteristic of a drug which is said to be as unique as a fingerprint.

Internal Standard

This standard is a deuterated version of the drug being tested and has similar characteristics with the one that is being tested. A precise amount is added before preparation so that the IS is prepared and analyzed similarly with the drug assayed. Quantification of the drug and qualification of the assay is done by comparing the signals produced by the internal standard with the signals produced of the drug being tested. The deuterated drug is made by substituting one or more hydrogen atoms with a deuterium atom. It exhibits similar characteristics as the non-deuterated drug but can be differentiated by the detection of the fragment mass.

Drugs are identified by the combination of its retention time by means of gas chromatography, fragmentation by means of mass spectrometry and specific information due to the presence of the IS

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REFERENCES

Heroin and Your Veins: The Incredibly Disgusting Story (Cobb, A.)


A Health Educator’s Guide to Understanding Drugs of Abuse Testing (Dasgupta, A.)


Evaluation of Saliva/Oral Fluid as an Alternate Drug Testing Specimen (Crouch et al.)


DRUG TESTS: THEIR USES AND LIMITATIONS (Tam Wai Ming)

URINALYSIS AND BODY FLUID (STRASINGER)

http://www.pbs.org/wnet/wideangle/episodes/bitter-harvest/from-poppy-to-heroin/step-1-planting-opium-poppy/3168/

http://www.streetdrugs.org/html%20files/Heroin.html


http://www.buzzle.com/articles/saliva-drug-test-facts.html


http://www.buzzle.com/articles/facts-about-hair-drug-test.html


http://www.buzzle.com/articles/hair-drug-testing-facts.html


http://kidshealth.org/kid/grow/drugs_alcohol/know_drugs_heroin.html


http://www.drugpolicy.org/drug-facts/heroin-facts


http://www.drugfree.org/drug-guide/heroin


http://www.drugfreeworld.org/drugfacts/heroin.html

http://www.accubiotech.com/templates/T_yestem_News/index.aspx?nodeid=49&page=ContentPage&contentid=212#3

http://www.cumberlandheights.org/addiction/heroin_recovery.aspx

http://www.meditests.com/urdrugofabte2.html

http://www.drugfreeworld.org/drugfacts/heroin/international-statistics.html

http://www.nlm.nih.gov/medlineplus/ency/article/003578.htm

http://www.norchemlab.com/wp-content/uploads/2011/10/Principle-of-GCMS.pdf

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Heroin Withdrawal and Treatment

 HEROIN WITHDRAWAL

Withdrawal from heroin abuse may cause abrupt withdrawal symptoms to the user causing him/her great discomfort. The first step in heroin addiction is detoxification. However, detox alone cannot completely treat heroin addiction. Sudden withdrawal from this addictive drug can be fatal for heavily dependent users who are in poor health.

WITHDRAWAL SYMPTOMS

When an addict stops heroin use, this drug will no longer act as an opiod receptor in the central nervous system. The body then tries to normalize brain function. Prolonged heroin use causes the body to become dependent to it and adapts to its presence. When heroin is suddenly reduced, withdrawal symptoms occur. Therefore, the more heroin is taken, worse withdrawal symptoms occur. Detoxification from heroin will make the body think that it is not working properly since it has already adapted to its presence which is why heroin withdrawal is difficult.

After 6-24 hours of not using heroin, withdrawal symptoms occur.These symptoms may include inner restlessness, chills, constipation, fever, malaise, nausea, and vomiting. Latent withdrawal symptoms include abnormal cramps, diarrhea, dilated pupils, disturbed sleep, depression and drug cravings. These symptoms will only subside a week after the last heroin use.

TREATMENT

Pharmaceutical therapy is also one option for treatment. This is done with the use of synthetic drugs such as methadone or buprenorphine which block the effects of heroin. 

Pharmaceutical therapy combined with rehabilitation programs is an effective means of treating a heroin addict. These may include family counseling, group therapy, 12-step program meetings, relapse prevention and aftercare programs. Persistent monitoring of heroin withdrawal and supportive care are important in preventing relapse.

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Hair Follicle Testing

          

Metabolites or circulating drugs may be detected in hair. First, these are deposited in the hair follicle and as the hair grows out, the metabolites will get trapped on the hair shaft and then in the keratin matrix. Drugs may also enter the hair through sweat, oil or the external environment. A 3-cm hair specimen may be able to indicate whether or not the person has been abusing drugs in the past 3 months. It is recommended that the hair specimen must be approximately 1.5 inches for drug abuse testing. When a person takes a drug, it takes approximately 4 to 5 days for the affected shaft to grow. However, there are a lot of factors that may affect the levels of the drug incorporated in a person’s hair. This means that high levels do not necessarily mean a more frequent drug abuse. It was demonstrated by Rothe et al that the doses of administered heroin and the measured concentration of opiates from the hair specimen were not correlated. The drug purity, metabolism, frequency of abuse and hair color are the factors that may affect the drug or metabolite amount incorporated in hair.

Natural hair color is an important factor in evaluating drug concentration. Drugs or metabolites are less incorporated in white fibers as compared with the pigmented ones. Melanin contains eumelanin which is accountable for hair with the colors black or brown whereas pheomelanin is responsible for red-colored hair. Drugs tend to bind less to pheomelanin than in eumelanin. Low abuse of drugs might not be detected in treated hair (e.g. bleached, permed or dyed). However, it can be detected in regular drug abuse but with reduced concentrations. Bleached or chemically-treated hair is more at risk to environmental drug uptake. Drugs already incorporated in hair are less removed in shampooing but this is effective in removing drugs absorbed from the environment. In hair follicle drug testing, heroin abuse can be detected as long as 90 days prior to testing.

COLLECTION AND TESTING

Hair sample is obtained and tested under the guidelines of the Society of Hair Testing. First, hair is thoroughly washed with the use of an organic solvent and with water subsequently. The washes can also be tested for the presence of the drug and its metabolites. After washing, the hair is cut into pieces and a small part is to be tested using Enzyme-linked Immunosorbent Assay (ELISA).

Quantity of hair needed

For this test, approximately 50 to 80 strands (45 mg) of hair are needed. There is slight variation due to hair color, thickness and texture. Samples obtained directly from hair comb or brush may not be used as samples should be taken live from the patient by cutting the hair strand as close to the scalp as possible.

ADVANTAGES

·         Hair follicle testing is reliable and accurate. It is considered to be better than urine testing as some drugs and their metabolites are easily flushed out of the bloodstream (3-7 days) whereas drugs trapped in the hair follicle for months to years.

·         It provides complementary information about the long-term drug abuse history of the patient.

·         Collection is non-invasive and the drugs incorporated in the hair have longer stability and they are bound for a long time.

·         Some people try to avoid hair follicle drug testing by going bald but avoidance is not possible since body hair can be used as a specimen as well.

·   The minimum concentration of metabolites that can be detected in hair follicle testing is low. Opiates trapped in hair with a concentration of only 100 pg/mg can be detected by using this test.

·         It can detect the following drugs: Cocaine (Cocaine & Benzoylecgonine), Marijuana, Opiates (Codeine, Morphine & 6-Monacteyl Morphine), Methamphetamine (Methamphetamine/Amphetamine & Ecstasy), and Phencyclidine (PCP).

DISADVANTAGES

·         Costly and time-consuming (minimum of 1 month)

·         It should be performed inside the laboratory

·         There are no available on-site testing kits

·         It cannot detect very recent drug abuse (1 to 7 days before testing)

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Oral Fluid Testing

Drug abuse testing may be performed using saliva as the specimen. Mixed saliva or “oral fluid” comes from the salivary glands, oral mucosa and gingival cervices. Drugs from the blood are transported to the saliva by means of passive diffusion. Within just a few minutes, the drugs and metabolites present in plasma are distributed to the salivary glands and are diffused into the saliva after parenteral administration. Therefore, the concentration of the drug in the saliva is correlated with the drug concentration in blood. Saliva is capable of retaining trace amounts of drugs and its metabolites for about 24-36 hours. However, Alain Verstraete from Yale University had reported that upon consumption of opiates, it only takes about 5 hours up to 48 hours for it to become undetectable in saliva. 

SPECIMEN COLLECTION

Collection of saliva/oral fluid is quick, easy and non-invasive. It does not require the presence of medically-trained personnel and it can also be tested at the collection site such as in point-of-care devices. There are several methods in which saliva can be collected.

1. Unstimulated Collection

• Draining Method

Performed by letting the saliva drip from the mouth freely into the specimen container

2. Stimulated Collection

• Without the use of an external stimulus

Tongue, cheek or lip movements

• Chemical Methods

Performed by chewing paraffin wax, gum base or chewing gum

• Lemon drop or Citric acid

A lemon drop is placed in the mouth to serve as a gustatory stimulus for the production of saliva

Disadvantages of stimulated saliva production

The use of parafilm may produce erroneous results since this absorbs some drugs. This may also interfere with chromatographic analyses producing inaccurate results.

The use of citric acid may alter saliva pH and its drug concentration.

The use of cotton balls may also change immunoassay results.

Collection

After stimulation, saliva can be spit, swabbed, or suctioned from the mouth. Collection may also be performed by using absorbent materials (e.g. cotton balls). Saliva will be extracted from the absorbent material via centrifugation or pressure application.

TESTING

                

Saliva drug testing kits

Principle: Competitive Binding Prinicple

The test device contains an absorbent pad wherein the sample that contains the drugs and its metabolites compete with the drug conjugate that is immobilized on a porous membrane for limited antibody sites. As the sample flows through the absorbent pad, the free drug that is contained in the saliva binds with the labeled antibody-dye conjugate now forming an antibody-antigen complex. This complex competes with the immobilized antigen conjugate located in the “test” zone and will not produce a colored band when the drug exceeds the detection level. A colored band is formed in the “control zone” when the unbound dye conjugate binds with the reagent indicating that the device is functioning properly.

Procedure:

Strip Test

1. Bring both the kit and specimen at room temperature.

2. Remove test strip from the pouch.

3. Dip the strip into the container with the arrows pointing towards it.

4. The saliva should exactly reach the maximum line (no more, no less).

5. Hold the saliva until a reddish color is seen at the lower edge of the strip (approximately 10 seconds).

6. Withdraw the strip and place on a clean, dry surface facing up.

7. Read results within the span of time indicated in test pack.

Cassette test

1. Bring the test kit and specimen in room temperature.

2. Remove cassette from the pouch.

3. Place kit on a flat, dry surface.

4. Use the dropper to dispense 3 drops of saliva into the sample well.

5. Start timing and read results within the span of time indicated in the test pack.

Interpretation of Results:

Positive – presence of only one (1) colored band in the “control” zone

Negative – two (2) colored bands, one in the “control” zone and one in the “test” zone

Invalid result – no colored band formation in the “control zone”

ADVANTAGES

·         Cheap cost and convenient

·         Results may be interpreted immediately (approximately 5 to 15 minutes).

·         Donor-friendly and ease of collection

·         It can detect 8 different drugs up to 5 or 6 types at a time.

·         Mouth swab testing is the best method in detecting recent drug abuse.

·         It does not require medically-trained personnel for its performance.

DISADVANTAGES

·   This method is not approved by the Substance Abuse and Mental Health Services Administration (SAMHSA) for use in Federal Mandated Drug Testing.

·         Most drugs do not remain present in the person’s saliva and may not be detected after 12-24 hours of consumption.

·         It is less sensitive in detecting THC and Cannabinoids.

·         More expensive than urine testing.

·         False positive results may be seen in patients taking in medicines such as Ibuprofen, Motrin, and Advil.

·         Inferior compared with hair follicle drug testing.

·         Detox drinks and smoking are very effective in masking the presence of the drug and its metabolites in saliva.

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Blood Testing

Aside from urine as the widely used specimen of choice for testing of heroin, blood can also be used in heroin determination because as it may not stay that long in the blood long as in urine, it will rather give a good correlation between the level of drug and its pharmacologic deficiency in the body.  Heroin stays in the blood up to 6hours.

Sample collection: A finger prick can be enough to use as a specimen sample but venipuncture may also be possible when further tests are needed. 

Blood Drug Testing kits

Principle: The principle of blood testing in Heroin is much similar to the principle used in urine and oral fluid. The test device kit uses competitive binding immunoassay where competition happens with the drug and/or drug metabolites compete with the immobilized drug conjugate having limited antibody sites. The blood sample then binds with the labelled antibody-dye conjugate to form an antigen-antibody complex. Since there is an antigen-antibody complex, when it reach the test zone it will not produce any colored band when it exceeds the detection level. Continuing to the control zone, the unbound dye conjugate binds with the reagent giving a colored band indicating the validity of the test kit. 

Interpretation of results:

Positive: Presence of colored band in the control zone and none in the test zone

Negative: Presence of colored band for both test and control zone

Invalid: Absence of colored band in the control zone even with or without the colored band in the test zone. Repetition of test is recommended.

Advantages: 

 - blood specimen will help detect particular amount or level of Heroin

 - it serves as a specimen for early detection unlike others

Disadvantages: 

- since Heroin can only be detected within 6hours in the blood, blood as a specimen of choice will give a false negative result if tested further time. 

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Urine Testing

Urine is the most widely used specimen for drugs of abuse testing because of the advantages of large specimen volume and relatively high drug concentrations that render drug detection comparatively easier than in other specimens. Drugs in urine are detectable by standard drug test on urine between 1-3days. Urine sample of individual is under tightly controlled condition.

The person will likely be asked to go to a testing facility where he or she will need to remove street clothing and put on a hospital gown. This ensures that a clean sample from someone else is not removed in to the testing center and substituted fort the subject's urine.

Once they changed into the gown, he or she is accompanied to the testing area. This is a washroom where the water in the toilet tank has been dyed so that an attempt to water down the sample by adding toilet water will be quite obvious. The water supply at the sink will have been shut off so that a person will not intended to use that source of liquid to alter the sample. A staff member will be waiting nearby to take the sample once it has been provided. The standard protocol is to test it to confirm that the temperature is consistent with normal body temperature.

A negative reading on drug test doesn’t mean that there is no presence of heroin in the body. According to Mandatory Guidelines for Federal Workplace Drug Testing Program, it simply means that sample taken didn't record a level higher than the threshold.


The cutoff value is 2000 nanograms per milliliter. It is usually repeated when the initial urine test is positive for drugs, including horse. If a second result have reach the cutoff value 2000 ng/ml means it is positive and the subject will be facing consequences, and none of them will be good.

ADVANTAGES

• It has the highest assurance of reliable results

• It is least expensive

• On-site testing kits are available

• Acceptable in court to withstand legal challenge

DISADVANTAGES

• It is exposed to adulteration or substitution

• It is does not need dose-concentration relationship

• The test are sometimes viewed as psychologically invasive or embarrassing

• Biological hazard for specimen handling and shipping to laboratory

Window of detection: Typically 1 to 3 days, except for cannabis (1 day to 2 weeks)

Urine Drug Specimen Collection Procedure

1. The collector must wash hands and wears gloves.

2. The collector must add bluing agent (dye) to the toilet water reservoir to stop from an adulterated specimen.


3. The collector must removes any source of water other than toilet by applying a tape to the the toilet lid and faucet handles.


4. The donor must give photo identification from employer representative.

5. The collector should complete the step 1 of the chain of custody (COC) form and has the donor sign the form.


6. The donor leaves his or her bag or  purse outside the collection area to avoid the contaminating the urine by the any substances.


7. The donor should washes his or her hands and receives a specimen cup.

8. The collector must observe for unauthorized water use. The collector must remains in the restroom but outside the stall, unless a witnessed collection is requested.


9. The donor hands specimen cup to the collector. Transfer is documented.


10. The collector checks the required amount volume, it should be 30–45 mL and for abnormal color of urine.


11. The collector must check the temperature strip on the specimen cup reads. It should be beyond 32.5C to37.7C. The collector records on the Chain of  (Step 2) form, the in-range temperature of the specimen . If the specimen temperature is out of range this means the specimen is questionable to have been diluted or adulterated. A new specimen must be collected and a supervisor notified.


12. The specimen must remain in the sight by the donor and collector at all times.


13. The collector leave the specimen identification strips from the

Chain of Custody form and attach them on the capped bottle, covering both sides of the cap.


14. The donor initials the specimen bottle seals.


15. The date and time are labelled on the seals.


16. The donor completes step 4 on the Chain of Custody form.


17. The collector completes step 5 on the  Chain of Custody form.


18. Each time the specimen is handled, transferred, or placed in storage, every individual must

be identified and the date and purpose of the change recorded.


19. The collector follows laboratory-specific instructions for packaging the specimen bottles

and laboratory copies of the Chain of Custody form.


20. The collector distributes the Chain of Custodycopies to appropriate personnel.

Urine Drug Test Kit for Heroin

Opiate (HEROINE, MORPHINE) Cassette Drug Urine Test Kit

It is simple, accurate and reliable. All tests are CE Marked and FDA Approved, with an accuracy level of 99%. It is easy to follow instructions.

PRINCIPLE OF THE TEST
The test is a competitive binding immunoassay in which drug and drug metabolites in a urine sample compete with immobilized drug conjugate for limited labeled antibody binding sites. By utilizing antibodies that are specific to different drug classes, the test permits independent, simultaneous detection of two drugs from a single sample. The approximate run time is 5 minutes. It's cutoff value is 2000 nanograms per milliliter.

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