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Medico-Legal Update

The use of saliva as an unconventional sample for drug detection: A review

Author(s): Shikha Shrivastava, Bastian TS, Anil Singh, Rohit Jaiswal

Vol. 8, No. 2 (2008-07 - 2008-12)

Shikha Shrivastava٭, Bastian TS٭٭, Anil Singh٭٭٭, Rohit Jaiswal٭٭٭٭

Department of Oral Pathology and Microbiology, Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow 226025


Global trends in drug trafficking and drug usage patterns indicate a continuing pattern of escalation throughout the world. Over the last two decades, urine analysis has evolved into a highly accurate means for determining whether individuals have been exposed to illicit drugs of abuse. Advances have also been made in the use of alternate biological matrices such as hair, oral fluids and sweat for drug testing. This review evaluates the use of saliva in drug analysis and in therapeutic and toxicological monitoring.

Keywords: saliva, drug detection, collection of saliva


Testing for drugs of abuse evolved in United States during the 1960s within the armed forces. The purpose of drug testing is to determine whether an individual has used any particular substance that might have affected their well-being or behaviour. The most common scenarios where testing is undertaken to detect psychoactive drug use include workplace drug testing, forensic drug testing (e.g. with drugs and driving) and compliance with treatment1.

Drug testing has undergone major advances, particularly over the last 10 years. The use of alternative specimens to blood or urine for establishing exposure to drugs has become a significant direction in clinical and forensic toxicology. These alternative specimens include hair, sweat and oral fluid. Oral fluid has been seen as a non-invasive alternative to blood but also as an alternative to urine when substitution or adulteration is suspected.

This review outlines the roles and applications of testing for drugs in oral fluid, describes the relative advantages and disadvantages of this form of testing and illustrates applications of oral fluid testing for specific drugs.

Oral Fluid as a Drug-Testing Specimen

Oral fluid, sometimes called “mixed saliva,” comes from three major and several minor salivary glands. Strictly speaking, oral fluid is the mixed saliva from the glands and other constituents present in the mouth.

Oral fluid contains plasma electrolytes such as potassium, sodium, chloride, and bicarbonate and many other plasma constituents, such as enzymes, immunoglobulins, and DNA. The total volume of oral fluid produced by an adult may be in excess of 1000 ml/day with typical flows of 0.05 ml/min while sleeping, 0.5 ml/min while spitting, and 1 ml/min to 3 ml/min while chewing gum2.

A number of drugs are known to affect the secretion of oral fluid. Most commonly these are amphetamines, including the designer forms such as ecstasy (MDMA), and cannabis. Other drugs include the sedating antihistamines, antipsychotic drugs, anticholinergic drugs and a number of antidepressants. There are less commonly used drugs that increase flow and these include clonidine, pilocarpine and beta-2 stimulants (salbutamol, terbutaline etc)2.

Many drugs of interest in the criminal justice setting have been detected in oral fluid, including ethanol, methamphetamine, amphetamine, barbiturates, benzodiazepines, heroin, cocaine, cannabinoids, marijuana, nicotine, opioids and phencyclidine3.

Mechanisms of drug transfer from blood to saliva

Landon and Mahmod (1982) suggested that the possible routes which may lead to a drug being present in mixed saliva are passive transcellular diffusion, ultrafiltration, active transport and pinocytosis. However, there is no evidence that pinocytosis plays any role4.

Mechanisms of salivary drug transport

Most drugs appear to enter saliva by a simple passive diffusion process which is characterized by the transfer of drug molecules down a concentration gradient. Paxton (1979) suggested that the rate of diffusion of a drug is a function of the concentration gradient, the surface area over which the transfer occurs, the thickness of the membrane, and a diffusion constant that depends on the physico-chemical properties of each drug (molecular weight, dissociation constants, lipid solubility, and protein binding)4. Although less common, low molecular mass compounds can also be transferred into oral fluid by active secretion or diffusion through pores in the membrane3.

Concentration of lipid-insoluble, non-electrolytes in saliva as a function of molecular size4.

Collection of saliva

A variety of methods are available for collecting saliva. Some involve stimulating saliva production, while others target collection of unstimulated or nonstimulated saliva. According to Navazesh (1993) unstimulated saliva can be collected by the draining method, which is performed by allowing saliva to drip from the mouth into a collection container. Several techniques may be used to collect stimulated saliva. The simplest involves tongue, cheek, or lip movements without the use of an external stimulus. Chewing paraffin wax, parafilm, teflon, rubber bands, gum base, or chewing gum are usually referred to as mechanical methods of stimulating saliva production. A lemon drop or citric acid can be placed in the mouth to provide a gustatory stimulus for saliva production5.

Following stimulation by one or more of these methods, saliva can be spit, suctioned, or swabbed from the mouth. Some collection techniques combine stimulation and collection of the saliva using absorbent materials such as cotton balls or cotton rolls. After the absorbent material becomes saturated with saliva, it is removed from the mouth and the saliva is extracted by centrifugation or by applying pressure to the material4.

There are several potential problems associated with stimulating saliva production. Chang (1976) showed that parafilm absorb some drugs and therefore, give erroneous results when saliva is tested for drugs or drug metabolites. Also, paraffin contains compounds that may affect chromatographic analyses—again affecting drug testing accuracy. Some salivary stimulants may change the salivary composition and, therefore, affect the saliva-drug concentration. For example, citric acid may change saliva pH and consequently alter drug concentrations in the saliva. Citric acid and cotton have also been shown to alter immunoassay drug test results4.

Several devices are commercially available for collecting saliva. Some devices are based on the collection techniques just discussed. They carry names such as Oral Diffusion Sink® (Shipley et al., 1992; Hold et al., 1996), Proflow SialometerTM (Jones, 1995), Orasure® (Gomez et al., 1994), and SalivetteTM` (Shipley et al., 1992). They have been advocated for saliva collection when testing for ethanol, steroids, and many other drugs6.

Analytical methodology for the measurement of drugs in saliva

Pre-analytical manipulations

Once the samples have been collected, it is important that they be properly stored unless analyses are to be performed immediately. Opinions differ as to the procedure to be followed, but most workers freeze the sample to -20°C, while some workers recommend centrifugation before freezing and others recommend centrifugation after thawing and prior to analysis. In forensic work in which saliva samples have been taken primarily for serological purposes, it is common practice to subject the sample and container to boiling water temperatures for 15-30 minutes prior to freezing. Only in cases in which the toxic material present in saliva is volatile or heatunstable would this treatment be expected to be deleterious to later analysis of such saliva samples7.

Most authors use the centrifugate, either by extracting the centrifugate with an organic solvent at a desirable pH or by using the centrifugate directly in the analytical process without sample pretreatment (Roth, Beschke, Jauch, Zimmer and Koss, 1981). Caddy (1984) has extensively reviewed the different methods of analysis of saliva4.

Immunological methods

Immunological methods of analysis have been widely used for monitoring drugs in saliva and other body fluids, mainly because of their relative simplicity of use, requiring little or no extractive operations, their application to large batch analyses, and their sensitivity. Paxton and Donald (1980) in their studies found that they do, however, suffer from disadvantages in that they do not always possess the specificity required for distinguishing metabolites from the parent drug.

Radioimmunoassay (RIA) techniques have been used especially for the analysis of hormones in saliva, such as estradiol, progesterone, testosterone, cortisol, and cortisone. Other drugs, which have been measured with RIA are for instance, cocaine, cannabinoids, haloperidol, theophylline and cotinine4. The use of the alternative non-radioactive immunological procedure, an enzyme multiplied immunoassay technique (EMIT®), is based on competitive protein binding using an enzyme as a label and an antibody as a specific binding protein. The enzyme activity is related to the amount of drug in the sample and is measured spectrophotometrically. Although this assay is very easy to use and requires no radiochemical facilities, it is not used very often for monitoring saliva levels. Sensitivity may be one major reason for this. One has to be careful to use citric acid in combination with the EMIT® assay because the enzyme glucose-6- phosphate dehydrogenase (G6PD) used in the EMIT® assay for ethosuximide and phenytoin is inhibited 38% by citric acid4. Another direct immunoassay which is not subject to the disadvantages associated with the use of radioisotopes in RIA is described by De Boever et al. (1990). They developed a chemiluminescence immunoassay (CIA) using isoluminol for the detection of estradiol in saliva. Cai, Zhu and Chen (1993) measured phenytoin in 1 ml saliva samples by fluorescence polarization (FPIA)4.

Chromatographic methods

Thin-layer chromatography (TLC) has the advantage of simplicity and allows the simultaneous determination of several samples. However, the major limiting factor is the detection of the respective spots on the thin-layer plate at low drug levels in saliva. Therefore, this technique is not often used in the drug monitoring of saliva. With the development of High-Performance TLC (HPTLC), Drehsen and Rohdewald (1981) tried to reach a higher sensitivity and precision. However, this procedure did not found wider application for saliva drug monitoring4.

The most popular analytical procedure for the measurement at the nanogram or picogram level is based on Gas Chromatography (GC), or, in the hyphenated mode, with Mass Spectrometry (GC/MS). Thompson et al. (1987) detected cocaine in saliva with GC/MS for 12 to 36 hours after administration. Gould et al. (1986) developed a GC/MS procedure for the measurement of salivary testosterone in female subjects, a striking application, because of the inefficiency of RIA4.

Doping drugs in saliva

The most frequently used biological specimen for the determination of drugs in doping control is urine, since only a non-invasively obtained sample is acceptable for routine collection. Yet, even the acceptability of urine sample is being disputed in view of the potential invasion of privacy, especially if a directly observed collection is advisable to prevent adulteration or substitution of sample. That happens, for instance, when athletes try to escape detection by using urine from someone else. Another major disadvantage of urine is the variability in the renal clearance of drugs and their metabolites, which is largely due to fluctuations in the flow rate and pH of urine. Not all drugs are excreted in the urine, for instance, the lipid soluble -blocking drugs tend to be rapidly eliminated by various metabolism systems in the liver. At present, saliva is not used as a biological fluid for doping control. Although a qualitative doping control mainly depends on the sensitivity of the assay, the usefulness of saliva needs to be explored here further7.

Advantages and disadvantages of oral-fluid drug testing

As an alternative matrix for drug testing, oral fluid offers some distinct advantages. The matrix is relatively clean and readily accessible for sampling. Collection is easy, non-invasive, and inexpensive. Sampling can be better supervised without invasion of privacy, reducing the opportunity for sample adulteration or substitution. Oral fluid testing also offers the possibility of direct comparison of unbound, pharmacologically active drug concentrations to pharmacodynamic effects3. However, the oral fluid drug testing is associated with certain disadvantages. These include variability of salivary flow and pH, and a shorter detection window than urine for active drugs. In addition, specimen collection can have a serious impact on analytical findings and smaller volumes of oral fluid are generally collected. Furthermore, there are few reports of drug concentration data to guide interpretation of oral fluid test results. Oral fluid drug concentrations often are lower than concentrations in traditional biofluids requiring new and sensitive analytical methodology. Certain drugs like, cannabis derivatives and benzodiazepines do not pass readily from blood into saliva while the concentration of some drugs is exceptionally high, eg. morphine and codeine, because of buccal absorption following smoking or snorting3.


Monitoring illicit drug use in drug treatment programs is traditionally performed by analysis of urine. In recent years, interest in other biological matrices as alternative drug-testing tools has grown, with oral fluid as one of the most promising new matrices. The use of oral fluid has been found to offer significant promise when detection of relatively recent use of drugs is sought in a non-invasive manner. Technological advances do allow on site detection of drugs, but there are technical issues in relation to collection of oral fluid and in the variability of drug concentrations (of different drug types) in this fluid. More research is needed to further the detection of drugs present in this fluid, which should allow improved reliability of detection of drugs. Similarly, future technological developments of onsite devices should allow more sensitive and reliable detection of a number of drugs.


  1. Cone EJ. Legal, workplace, and treatment drug testing with alternate biological matrices on a global scale. Forensic Science International 2001; 121 (1); 7-15.
  2. Aps JK, Martens LC. Review: The physiology of saliva and transfer of drugs into saliva. Forensic Sci Int 2005; 150: 119-131.
  3. Dams R, Choo RE, Lambert WE, Jones H, Huestis MA. Oral Fluid as an Alternative Matrix to Monitor Opiate and Cocaine use in Substance Abuse Treatment Patients. Drug Alcohol Depend 2007; 87(2-3): 258–267.
  4. Hold KM, de Boer D, Zuidema J, Maes RAA. Saliva as an Analytical Tool in Toxicology. Int. J of Drug Testing 1995; 1(1): 1-36.
  5. Navazash H. Methods for collecting saliva. Ann NY Acad Sci. 1993; 694: 72-77.
  6. Drummer OH. Drug Testing in Oral Fluid. Clin Biochem Rev 2006; 27: 117-129.
  7. Rai B: Oral Fluid in Toxicology. The Internet Journal of Toxicology; 2007: 3(2).
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