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

The most common routes for a drug to migrate to saliva are passive transcellular diffusion, ultrafiltration, active transport and passive diffusion.



#### **Table 1.**

*Factors influencing passive diffusion of a drug from blood to saliva.*

constant that depends on the physico-chemical properties of each drug [15]. The variables which influence this type of transport are listed in **Table 1** (Landon and Mahmod, 1982) [16].

Salivary secretion is a reflex response controlled by both parasympathetic and sympathetic secretomotor nerves. This is an important factor influencing oral fluid availability and potential drug concentrations. Taking medication which affects either the central nervous system or the peripheral nervous system (or medication which mimics the latter as a side effect) alters salivary composition and salivary volume. Therefore, patients suffering from systemic diseases may show alterations in salivary gland secretion and electrolyte concentrations. Finally, diet and age also have an impact on composition and volume of saliva [8].

### **3. Methods and techniques**

#### **3.1 The sampling**

It is essential to prepare Standard Operating Procedures (SOPs) relating to the collection and storage of the oral fluid sample, as well as the training of personnel assigned to take and ship the sample to the laboratory where the toxicological analysis will be carried out. It follows that it is essential to document:

• respect for the privacy and security of the person undergoing analytical assessment;

**139**

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

identity records of the personnel authorised to handle it.

• that the informed consent form has been completed in its entirety by the

person undergoing the analytical assessment (unless there is a formal mandate

• the use of particular medicines that may interfere with the analytical results;

• the traceability of the sample through appropriate records of its movement, from the place of sampling to the laboratory that receives it, including the

Neat oral fluid can be collected from expectoration (or spitting), but this is relatively viscous and can therefore be challenging to work with and analyse in the laboratory. It may also be contaminated with food and oral debris, which makes centrifugation essential. In addition, sensitive detection techniques are required, because the volume collected will often be less than 1 mL. Normally, the absorbent foam swab or pad used to collect the oral fluid is added to a diluent. After mixing, the solution is ready for drug analysis. Other devices involve squeezing absorbed oral fluid from a pad or foam directly onto the drug-detection device, a process that can take one to three minutes. A number of devices incorporate some form of indicator to show when an adequate amount of oral

A number of drugs affect the secretion of oral fluid [8], mostly cannabis and amphetamines, including designer drugs such as MDMA. Other drugs include the sedating antihistamines, antipsychotic drugs, anticholinergic drugs and several antidepressants. Less commonly used drugs increase saliva flow and these include clonidine, pilocarpine and beta-2 stimulants (salbutamol, terbutaline, etc). Overall, there is significant intra- and inter-subject variation in relation to drug concentrations depending on the technique used, the physiology of the person and the factors

An important aspect to consider is the choice of analytical technique used for the detection of drugs and metabolites in saliva. A fundamental element is the certainty and reliability of the results, from both qualitative and quantitative perspectives. The results of quantitative determination, though, are not easy to interpret as the information that makes it possible to trace the metabolic process is often unavailable (e.g. time the drug was taken, amount of active ingredient, and route of administration). In many forensic contexts, oral fluid is analysed with screening methods. The semiquantitative results obtained must be validated by confirmatory techniques, such as liquid chromatography combined with mass spectrometry [18]. Oral fluids (OF) have been recently introduced as a biological matrix useful for roadside testing to determine illicit drug use because the time course of drugs in oral fluid may resemble that of plasma. Moreover, OF can be considered a valid alternative specimen for confirmation testing because drugs are excreted in saliva mainly as parent compounds [19–21]. In fact police officers, without medical supervision, are not authorised to employ invasive methods but they can collect OF samples. A very comprehensive review of the analysis of drugs of abuse in OF was conducted by Reinstadler et al. [22]. Other studies [23] have highlighted the importance of both the sample treatment process and the use of hyphenated instruments in obtaining analytical performances that satisfy current regulations in terms of sensitivity,

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

from the Legal Authority);

fluid has been collected [17].

**3.2 Analytical techniques**

affecting drug concentration in oral fluid.

selectivity and fast confirmatory analysis.


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

constant that depends on the physico-chemical properties of each drug [15]. The variables which influence this type of transport are listed in **Table 1**

*RELATING TO THE CIRCULATING DRUG LEVEL IN THE FREE (NONPROTEIN-BOUND) FORM*

Salivary secretion is a reflex response controlled by both parasympathetic and sympathetic secretomotor nerves. This is an important factor influencing oral fluid availability and potential drug concentrations. Taking medication which affects either the central nervous system or the peripheral nervous system (or medication which mimics the latter as a side effect) alters salivary composition and salivary volume. Therefore, patients suffering from systemic diseases may show alterations in salivary gland secretion and electrolyte concentrations. Finally, diet and age also

It is essential to prepare Standard Operating Procedures (SOPs) relating to the collection and storage of the oral fluid sample, as well as the training of personnel assigned to take and ship the sample to the laboratory where the toxicological

• respect for the privacy and security of the person undergoing analytical

analysis will be carried out. It follows that it is essential to document:

• the identity of the person undergoing analytical assessment;

• the location where the sample of oral fluid has been collected;

• that no falsification or tampering of the sample has taken place;

(Landon and Mahmod, 1982) [16].

*Factors influencing passive diffusion of a drug from blood to saliva.*

*RELATING TO DRUG* Lipid-solubility

Charged or neutral *RELATING TO SALIVA*

Saliva pH Saliva flow rate

**Table 1.**

Acidic or basic, and the pKa

Dose and clearance of drug Nonprotein-bound blood level

Molecular weight and spatial configuration

Saliva-binding proteins - usually minimal

Enzymes in saliva capable of metabolising the drug

**3. Methods and techniques**

**3.1 The sampling**

assessment;

have an impact on composition and volume of saliva [8].

**138**


Neat oral fluid can be collected from expectoration (or spitting), but this is relatively viscous and can therefore be challenging to work with and analyse in the laboratory. It may also be contaminated with food and oral debris, which makes centrifugation essential. In addition, sensitive detection techniques are required, because the volume collected will often be less than 1 mL. Normally, the absorbent foam swab or pad used to collect the oral fluid is added to a diluent. After mixing, the solution is ready for drug analysis. Other devices involve squeezing absorbed oral fluid from a pad or foam directly onto the drug-detection device, a process that can take one to three minutes. A number of devices incorporate some form of indicator to show when an adequate amount of oral fluid has been collected [17].

A number of drugs affect the secretion of oral fluid [8], mostly cannabis and amphetamines, including designer drugs such as MDMA. Other drugs include the sedating antihistamines, antipsychotic drugs, anticholinergic drugs and several antidepressants. Less commonly used drugs increase saliva flow and these include clonidine, pilocarpine and beta-2 stimulants (salbutamol, terbutaline, etc). Overall, there is significant intra- and inter-subject variation in relation to drug concentrations depending on the technique used, the physiology of the person and the factors affecting drug concentration in oral fluid.

#### **3.2 Analytical techniques**

An important aspect to consider is the choice of analytical technique used for the detection of drugs and metabolites in saliva. A fundamental element is the certainty and reliability of the results, from both qualitative and quantitative perspectives. The results of quantitative determination, though, are not easy to interpret as the information that makes it possible to trace the metabolic process is often unavailable (e.g. time the drug was taken, amount of active ingredient, and route of administration).

In many forensic contexts, oral fluid is analysed with screening methods. The semiquantitative results obtained must be validated by confirmatory techniques, such as liquid chromatography combined with mass spectrometry [18]. Oral fluids (OF) have been recently introduced as a biological matrix useful for roadside testing to determine illicit drug use because the time course of drugs in oral fluid may resemble that of plasma. Moreover, OF can be considered a valid alternative specimen for confirmation testing because drugs are excreted in saliva mainly as parent compounds [19–21]. In fact police officers, without medical supervision, are not authorised to employ invasive methods but they can collect OF samples. A very comprehensive review of the analysis of drugs of abuse in OF was conducted by Reinstadler et al. [22]. Other studies [23] have highlighted the importance of both the sample treatment process and the use of hyphenated instruments in obtaining analytical performances that satisfy current regulations in terms of sensitivity, selectivity and fast confirmatory analysis.

#### *3.2.1 On-site screening test*

Recent data have shown improvements in the effectiveness of on-site drug testing using oral fluid, and significant progress has been made in terms of sample collection and accuracy of analysis [24].

A number of field drug testing devices are available and used in many countries to perform on-site testing on oral fluids in the context of Driving Under the Influence of Drugs (DUID) [25]. For example, DrugWipe® is an immunochromatographic test strip, based on the Frontline urine test strip from Boehringer Mannheim. A pink colour in the test window indicates the presence of the analyte in question, but different devices are normally required to detect the various classes of drugs of abuse. However, a recent version of this device, DrugWipe 5A, is capable of indicating the simultaneous use of cannabis, amphetamine, methamphetamine, ecstasy, cocaine, and opiates [26]. A recent study investigated the reliability of DrugWipe 5A in establishing exposure to principal drugs of abuse (cannabis, amphetamines, cocaine, and opiates) using oral fluid specimens by comparing the on-site results with headspace solid-phase microextraction (HS-SPME) gas chromatography–mass spectrometry (GC–MS) analyses on extractions from the sample collection pad [27].

Another point of collection test, Rapid STAT®, has broken new ground by combining the convenience of oral fluid collection, surface wipe testing or pure substance measurements with the sensitivity, accuracy and precision of a laboratory based test, with speedy results (in a few minutes).

**Table 2** shows the recommended minimum detectable concentrations of drugs in oral fluid according to SAMHSA and European Union roadside assessment testing study (ROSITA) cut-off levels [28].

#### *3.2.2 Laboratory screening test*

The enzyme-linked immunosorbent assay (ELISA) is a sensitive and versatile test used in many fields to detect and measure substances in biological samples (**Figure 2**). For almost 50 years it has remained a trusted testing technique for everything from food allergen detection to medical screening for various illnesses. For the toxicology market specifically, ELISA is an excellent and cost-effective solution which meets high-throughput screening (HTS) needs. The procedure is simple and easily automated or it can be conducted by a laboratory technician. It basically works around the principle of competition between two substances in a given sample: an enzyme conjugate such as horseradish peroxidase (HRP) is used to compete with a target substance for a limited number of specific binding sites on a precoated microplate.


#### **Table 2.**

*Recommended minimum detectable concentrations of drugs in oral fluid – Instrumental devices field testing.*

**141**

tography changes.

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

azepines despite their low oral fluid concentrations [33].

*3.2.3 Confirmatory analysis*

*Fully automated Elisa analyser.*

**Figure 2.**

The different available types of ELISAs provide a reliable means for screening oral fluid. In general these work adequately for amphetamines [29], buprenorphine, cocaine [30], methadone [31], and other opioids [32]. Cannabis may pose more difficulties, particularly if the immunoassay has little cross-reactivity to *tetrahydrocannabinol* (THC), the main psychoactive component of the drug. Even so, enzyme immunoassay has been successfully used for cannabis; the same applies to benzodi-

Confirmatory techniques for drugs in oral fluid [20] are mostly adapted from those used in the analyses of blood or plasma/serum specimens. Recovery of drugs is not typically a limiting factor, considering the higher water content and lower protein levels of oral fluid compared to blood. However, the sample volume of oral fluid will be smaller, with potentially lower concentrations, which means that more adjustments are required to analytical techniques. Indeed, in saliva analysis the detection or quantification limit for drugs is very much determined by the type of screening test and its application. The confirmation method must be able to produce an analytical result that is optimally independent from that of the screening. Therefore, it must be based on different physico-chemical principles and have superior analytical selectivity and sensitivity. In this regard, a quantitative confirmatory method capable of reaching a lower limit of quantification (LLOQ ) equal to at least half the cut-off of the screening method is considered acceptable. The use of a confirmatory method which is based on the measurement of a similar analytical signal is not acceptable since it is highly correlated to that of the screening (e.g. confirmation of a given immunochemical with another immunochemical method). The use of an identical chromatographic technique to confirm a set of data obtained by chromatography is acceptable if the detection technique combined with chroma-

The use of a chromatographic technique to confirm screening data obtained by chromatography with the same detection system is allowed only if the two separation techniques produce poorly correlated results (for example, two series of significantly different retention times, with the use of columns of different polarity or selectivity, etc). However, in the forensic toxicological field, chromatographic separation is always necessary in a confirmatory method; the general consensus of the international scientific community is that mass spectrometry (MS) with its many methodological possibilities can be combined with a chromatographic separation technique such as gas chromatography (GC), high pressure liquid chromatography (HPLC) or capillary electrophoresis (EC) for confirmatory analysis (**Figure 3**). Many methods

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

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

**Figure 2.** *Fully automated Elisa analyser.*

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

Recent data have shown improvements in the effectiveness of on-site drug testing using oral fluid, and significant progress has been made in terms of sample

A number of field drug testing devices are available and used in many countries to perform on-site testing on oral fluids in the context of Driving Under the Influence of Drugs (DUID) [25]. For example, DrugWipe® is an immunochromatographic test strip, based on the Frontline urine test strip from Boehringer Mannheim. A pink colour in the test window indicates the presence of the analyte in question, but different devices are normally required to detect the various classes of drugs of abuse. However, a recent version of this device, DrugWipe 5A, is capable of indicating the simultaneous use of cannabis, amphetamine, methamphetamine, ecstasy, cocaine, and opiates [26]. A recent study investigated the reliability of DrugWipe 5A in establishing exposure to principal drugs of abuse (cannabis, amphetamines, cocaine, and opiates) using oral fluid specimens by comparing the on-site results with headspace solid-phase microextraction (HS-SPME) gas chromatography–mass spectrometry (GC–MS) analyses on extractions from the sample

Another point of collection test, Rapid STAT®, has broken new ground by combining the convenience of oral fluid collection, surface wipe testing or pure substance measurements with the sensitivity, accuracy and precision of a laboratory

in oral fluid according to SAMHSA and European Union roadside assessment

**Table 2** shows the recommended minimum detectable concentrations of drugs

The enzyme-linked immunosorbent assay (ELISA) is a sensitive and versatile test used in many fields to detect and measure substances in biological samples (**Figure 2**). For almost 50 years it has remained a trusted testing technique for everything from food allergen detection to medical screening for various illnesses. For the toxicology market specifically, ELISA is an excellent and cost-effective solution which meets high-throughput screening (HTS) needs. The procedure is simple and easily automated or it can be conducted by a laboratory technician. It basically works around the principle of competition between two substances in a given sample: an enzyme conjugate such as horseradish peroxidase (HRP) is used to compete with a target substance for a limited number of specific binding sites on a

*Drug SAMHSA cut-offs (ng/mL) ROSITA cut-offs (ng/mL)*

*-tetrahydrocannabinol.*

Cocaine 00 5–10 Morphine 40 — 6-AM 4 10 Methamphetamine/Amphetamine/MDMA 50 70–90 THC CM 1.9

*Recommended minimum detectable concentrations of drugs in oral fluid – Instrumental devices field testing.*

*6-AM = 6-Acetylmorphine, MDMA = methylenedioxymethamphetamine, THC =* Δ*<sup>9</sup>*

*3.2.1 On-site screening test*

collection pad [27].

collection and accuracy of analysis [24].

based test, with speedy results (in a few minutes).

testing study (ROSITA) cut-off levels [28].

*3.2.2 Laboratory screening test*

precoated microplate.

**140**

**Table 2.**

The different available types of ELISAs provide a reliable means for screening oral fluid. In general these work adequately for amphetamines [29], buprenorphine, cocaine [30], methadone [31], and other opioids [32]. Cannabis may pose more difficulties, particularly if the immunoassay has little cross-reactivity to *tetrahydrocannabinol* (THC), the main psychoactive component of the drug. Even so, enzyme immunoassay has been successfully used for cannabis; the same applies to benzodiazepines despite their low oral fluid concentrations [33].
