**1. Introduction**

Detecting the presence of drugs or their metabolites in biological material requires different approaches and methods, depending on the purpose of the investigation and specific legal requirements. In the forensic toxicology field, multiple biological matrices are commonly used as diagnostic tools (such as blood, urine, keratin matrices, oral fluid, etc.) and the respective results, either alone or in combination with each other, provide useful elements for a correct diagnosis. An investigation may be prompted by various concerns: suitability to drive, professional driver suitability, employee and work suitability, suitability for gun permit, suitability for specific competition and/or contractual rules, diagnosis of use/abuse (also in the contexts of custody of minors and international adoptions), diagnosis of drug addiction, and diagnosis of intoxication in living or dead people.

Technical choices are based on these premises and purposes. For example, urine testing can typically determine the "recent" consumption of substances of abuse (with a temporal detection window of hours or even days depending on the pharmacokinetic characteristics of the substance in question). This sample can also be used to determine chronic drug use if the analysis is extended to several samples collected on different days and "by surprise" (i.e. with the shortest possible notice given to the interested party, not exceeding 24 hours). Chronic use, as well as previous patterns of use/abuse, can be verified by analysis of the hair matrix too.

In cases where it is necessary to quickly evaluate degree of substance intoxication (for example in an emergency situation) blood testing is particularly useful. Even so, over the past few years oral fluid has been increasingly studied as an alternative matrix of choice, and a number of reviews and papers have recently focused on various aspects of drug testing using oral fluid, although it has a shorter detection window than blood (**Figure 1**). Consideration should be given to the importance of oral fluid as a clinical diagnostic [1] and forensic tool and its relevance for a range of applications including workplace drug testing [2], drug driving [3], legal issues associated with drug testing [4], pharmacokinetics of selected drugs [5], and therapeutic drug monitoring (TDM) [6].

Regarding its composition, saliva is a very dilute fluid. Its major constituent is water (> 97%); other components include electrolytes, immunoglobins, enzymes and proteins. In normal conditions, healthy adults produce approximately 500–1500 mL saliva in 24 hours through the submandibular gland (about 65%), the parotid gland (23%) and the sublingual gland (4%), along with many other small glands distributed in the oral cavity (about 8%). Products of the salivary glands can be classified into four major components with different functions: mucus that serves as a lubricant; amylase, an enzyme that initiates the digestion of starch; lingual lipase, an enzyme that begins the fat digestion process; and a slightly alkaline electrolyte solution that moistens food so that it can be swallowed easily.

The most abundant salivary electrolytes are sodium, potassium, chloride and bicarbonate, while calcium, magnesium and phosphate are present in lesser concentrations. Other salivary constituents include substances transported from the blood through the gland into saliva [7].

Salivation can be stimulated or reduced by several factors. Electrolyte concentrations and volume of saliva produced are influenced by the time of day and type of salivation stimulus. In fact, the volume and composition of oral fluid can vary during the day and over time in each individual. Therefore, it can be said that its composition varies continuously, both quantatively and qualitatively [8]. When salivary constituents need to be identified, it should be emphasised that the results will depend on the subject's cooperation, psychological status, medication use, method of sampling and time of day.

Saliva has a slightly more acidic pH (6 to 7) than that of blood, and therefore all lipophilic psychoactive substances, with a weak basic nature, low molecular weight and blood protein binding of less than 50%, are preferentially excreted in saliva by passive diffusion of the free fraction of the substance in its ionised form. Moreover, the pH of saliva can change from being slightly acidic at rest, to basic (pH 8) at

**137**

mechanism.

*Salivary Analysis for Medico-Legal and Forensic Toxicological Purposes*

ultimate stimulation. Amylase and mucus also increase in concentration after

The first guidelines for the analysis of substances of abuse in saliva were proposed in 2004 in the United States by the "Substance Abuse and Mental Health Service Administration" (SAMHSA) [9] and were mainly intended for analyses carried out in the workplace to determine the possible use of substances. Subsequently, the "European Workplace Drug Testing Society" (EWDTS) [10] also drafted European guidelines, again oriented to analyses in workplaces. SAMHSA published its final Mandatory Guidelines for Federal Workplace Drug Testing Programs using Oral Fluid on October 25, 2019 in the Federal Register [11]. The new regulations only apply to federal workplaces, at the time of writing, but the

The most common routes for a drug to migrate to saliva are passive transcellular

through the capillary wall, basement membrane and acinar cell of the secretory end-piece, with the lipid layer of the epithelial cell wall providing the ratelimiting barrier. The same mechanism would probably enable these molecules to pass through the cells lining the ducts of the gland. The salivary concentrations of the lipid-soluble, unconjugated steroids such as oestriol, cortisol and testosterone approximate the unbound plasma concentrations. But, the concentration of the lipid-insoluble, conjugated steroid dehydroepiandrosterone sulphate is approximately 1% of the unbound plasma concentration [12].

a.**Passive transcellular diffusion:** highly lipid-soluble substances may pass

b.**Ultrafiltration (or paracellular transport)**: small polar molecules such as glycerol and sucrose enter saliva. The saliva/plasma (S/P) ratios of several small polar, lipid-insoluble compounds are plotted as a function of their molecular weight (MW). This mechanism is restricted to compounds with a MW of less than about 300 Da, and even those with a MW of about 150 Da are only filtered to a minimal extent. Furthermore, the flow rate of saliva should

c.**Active transport mechanism:** clearly operates for many electrolytes and for some proteins such as IgA. This mechanism has also been proven for some drugs. Lithium (MW = 7 Da) would be expected to appear in saliva by ultrafiltration. However, the findings of a S/P ratio of more than two indicates an active secretory mechanism [13]. Borzelleca (1965) [14] investigated whether penicillin and tetracycline were secreted in saliva. The secretion of these antibiotics in saliva appeared to be dependent on the concentration in the blood. Since the secretion of penicillin by the salivary apparatus and by the kidney were both inhibited by probenecid, an inhibitor of the active renal pathway, at least a part of the penicillin secretion in saliva involved an active

d.**Passive diffusion process**: is characterised by the transfer of drug molecules down a concentration gradient with no expenditure of energy. 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

impact is sure to reach beyond the initial scope of these regulations.

diffusion, ultrafiltration, active transport and passive diffusion.

not affect S/P ratios if diffusion is rapid and passive.

*DOI: http://dx.doi.org/10.5772/intechopen.95625*

**2. Drug transfer from blood to saliva**

stimulation [8].

**Figure 1.** *Drug detection times in different matrices.*

*Salivary Analysis for Medico-Legal and Forensic Toxicological Purposes DOI: http://dx.doi.org/10.5772/intechopen.95625*

*Forensic Analysis - Scientific and Medical Techniques and Evidence under the Microscope*

ous patterns of use/abuse, can be verified by analysis of the hair matrix too.

therapeutic drug monitoring (TDM) [6].

through the gland into saliva [7].

method of sampling and time of day.

*Drug detection times in different matrices.*

solution that moistens food so that it can be swallowed easily.

be used to determine chronic drug use if the analysis is extended to several samples collected on different days and "by surprise" (i.e. with the shortest possible notice given to the interested party, not exceeding 24 hours). Chronic use, as well as previ-

In cases where it is necessary to quickly evaluate degree of substance intoxication (for example in an emergency situation) blood testing is particularly useful. Even so, over the past few years oral fluid has been increasingly studied as an alternative matrix of choice, and a number of reviews and papers have recently focused on various aspects of drug testing using oral fluid, although it has a shorter detection window than blood (**Figure 1**). Consideration should be given to the importance of oral fluid as a clinical diagnostic [1] and forensic tool and its relevance for a range of applications including workplace drug testing [2], drug driving [3], legal issues associated with drug testing [4], pharmacokinetics of selected drugs [5], and

Regarding its composition, saliva is a very dilute fluid. Its major constituent is water (> 97%); other components include electrolytes, immunoglobins, enzymes and proteins. In normal conditions, healthy adults produce approximately 500–1500 mL saliva in 24 hours through the submandibular gland (about 65%), the parotid gland (23%) and the sublingual gland (4%), along with many other small glands distributed in the oral cavity (about 8%). Products of the salivary glands can be classified into four major components with different functions: mucus that serves as a lubricant; amylase, an enzyme that initiates the digestion of starch; lingual lipase, an enzyme that begins the fat digestion process; and a slightly alkaline electrolyte

The most abundant salivary electrolytes are sodium, potassium, chloride and bicarbonate, while calcium, magnesium and phosphate are present in lesser concentrations. Other salivary constituents include substances transported from the blood

Salivation can be stimulated or reduced by several factors. Electrolyte concentrations and volume of saliva produced are influenced by the time of day and type of salivation stimulus. In fact, the volume and composition of oral fluid can vary during the day and over time in each individual. Therefore, it can be said that its composition varies continuously, both quantatively and qualitatively [8]. When salivary constituents need to be identified, it should be emphasised that the results will depend on the subject's cooperation, psychological status, medication use,

Saliva has a slightly more acidic pH (6 to 7) than that of blood, and therefore all lipophilic psychoactive substances, with a weak basic nature, low molecular weight and blood protein binding of less than 50%, are preferentially excreted in saliva by passive diffusion of the free fraction of the substance in its ionised form. Moreover, the pH of saliva can change from being slightly acidic at rest, to basic (pH 8) at

**136**

**Figure 1.**

ultimate stimulation. Amylase and mucus also increase in concentration after stimulation [8].

The first guidelines for the analysis of substances of abuse in saliva were proposed in 2004 in the United States by the "Substance Abuse and Mental Health Service Administration" (SAMHSA) [9] and were mainly intended for analyses carried out in the workplace to determine the possible use of substances. Subsequently, the "European Workplace Drug Testing Society" (EWDTS) [10] also drafted European guidelines, again oriented to analyses in workplaces. SAMHSA published its final Mandatory Guidelines for Federal Workplace Drug Testing Programs using Oral Fluid on October 25, 2019 in the Federal Register [11]. The new regulations only apply to federal workplaces, at the time of writing, but the impact is sure to reach beyond the initial scope of these regulations.
