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 Table of Contents  
Year : 2016  |  Volume : 8  |  Issue : 1  |  Page : 18-22  

Laboratory approach for diagnosis of toluene-based inhalant abuse in a clinical setting

National Drug Dependence Treatment Centre, All India Institute of Medical Sciences, New Delhi, India

Date of Submission15-Mar-2015
Date of Decision03-Jun-2015
Date of Acceptance16-Jun-2015
Date of Web Publication13-Jan-2016

Correspondence Address:
Raka Jain
National Drug Dependence Treatment Centre, All India Institute of Medical Sciences, New Delhi
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0975-7406.164293

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The steady increase of inhalant abuse is a great challenge for analytical toxicologists. This review describes an overview of inhalant abuse including the extent of the problem, types of products abused, modes of administration, pharmacology and effects of inhalants, the role of laboratory, interpretation of laboratory results and clinical considerations. Regular laboratory screening for inhalant abuse as well as other substance abuse and health risk behaviors must be a part of standard clinical care.

Keywords: Analytical issues, hippuric acid, inhalant abuse, toluene

How to cite this article:
Jain R, Verma A. Laboratory approach for diagnosis of toluene-based inhalant abuse in a clinical setting. J Pharm Bioall Sci 2016;8:18-22

How to cite this URL:
Jain R, Verma A. Laboratory approach for diagnosis of toluene-based inhalant abuse in a clinical setting. J Pharm Bioall Sci [serial online] 2016 [cited 2022 Nov 30];8:18-22. Available from:

In the last few decades, inhalant abuse is on the rise and has become an issue of great concern. It is a serious drug problem worldwide, particularly in disadvantaged populations and among adolescents.[1],[2],[3],[4] Inhalant use is among the most prevalent, pernicious, and poorly understood forms of substance use.[5],[6] The reason of the abuse is that inhalants are easily available, relatively inexpensive and legally distributed which makes them accessible to children, adolescents, and the general population.[7] Moreover, use of these substances by inhalation allows their rapid and high concentrations in the lungs and the brain, with quick initiation of the effects.[6] Literature suggests that inhalant using individuals have greater antisocial attitudes, personal and familial dysfunction, peer deviance, suicidality, and substance abuse than their noninhalant-using counterparts.[8]

As the substance of abuse, inhalants are breathable chemical vapors that users intentionally inhale to produce a mind-altering “buzz” or high. Inhalant use increases the risk of numerous serious medical illnesses, even death.[9] Inhalants are one of the least-studied or discussed groups of abused substances.[5],[6],[10] Little is known about the prevalence and characteristics associated with progression to abuse and dependence among inhalant users.[11] The diagnosis of inhalant abuse relies almost entirely on a high index of suspicion. A diligent history and a thorough physical examination are the mainstays of diagnosis. More challenging is the management of inhalant abuse, which is known to cause extensive medical, psychiatric, and psychological damage, but there are hardly any pharmacological or nonpharmacological interventions available to tackle this problem.[12],[13],[14] Thus, diagnosis of inhalant use is very important and cannot be achieved without acquiring laboratory services. The purpose of the present review is to enlighten the role of laboratory for the treatment of an inhalant user in a clinical setting.

   Types of Products Abused Top

The substances inhaled are often common household products that contain volatile solvents, hair spray or aerosols, glue adhesives, shoe polish, gasoline, spray paints or solvents, marker pens, correction fluid, butane lighter fluid, propane gas, cooking sprays and household cleaners.[15] Some of the volatile substances in these products include toluene, chloroform, propane, acetone and many halogenated hydrocarbons. In India, ink eraser fluid or adhesives are most commonly used a form of inhalant.[16],[17],[18],[19],[20],[21] The main volatile constituent of eraser fluid or adhesives is toluene.

   Mode and Signs and Symptoms of Inhalant Abuse Top

Abusers may inhale vapors directly from a container (sniffing), from a bag into which a substance has been placed (bagging), or from a rag soaked with a substance and then placed over the mouth or nose (huffing).[2],[22]

Inhalant abuse can be detected by a clinical signs and behavior changes, but the effect varies among individuals.[6] Once inhaled, due to the lipophilic nature of toluene, it gets rapidly absorbed and act directly on the nervous system to produce mind-altering effects. Therefore, entry into the brain is extremely fast, and the onset of effects is almost immediate. Thus, within seconds, the user experiences intoxication and other effects similar to those from alcohol.[7]

Patients who have been abusing inhalants may report dizziness, irritability, tiredness, loss of appetite, headache, photophobia, or cough.[6],[23] Most symptoms are nonspecific and can be mistaken for those of other illnesses or syndromes. Signs of recent inhalant abuse include paint or oil stains on clothing or skin, spots or sores around the mouth, bloodshot eye, rhinorrhea, chemical odor on the breath, and a dazed appearance.[22] Patients with long-term inhalant abuse can present to the emergency department or office setting with a wide range of neuropsychiatric signs and symptoms. The most commonly recognized acute presentation is sudden unconsciousness or death during known inhalation of a solvent. Other, nonspecific complaints include: Memory loss, especially loss of short-term memory; diminished intelligence; delusions or hallucinations; slurred or changed speech; staggering, stumbling, or wide-based ataxic gait; visual and optical changes, such as nystagmus and hearing loss or sense of smell.[24],[25] Chronic inhalant abuse can damage cardiac, renal, hepatic, neurologic systems and bone marrow. Inhalant abuse during pregnancy can cause fetal abnormalities. Moreover, inhalant use can result in chemical and thermal burns,[26] withdrawal symptoms,[27] persistent mental illness,[28] and catastrophic medical emergencies such as ventricular arrhythmias leading to “sudden sniffing death."[29],[30] Unfortunately, no specific syndromes or clinical presentations confirm the inhalant abuse. Diagnosis of inhalant abuse is difficult and relies almost entirely on a thorough history and a high index of suspicion.[25]

   Pharmacology and Effects of Inhalants Top

Inhalants are readily absorbed through the lungs, with immediate and brief effects, and then relatively rapidly metabolized predominately through the cytochrome P450 (CYP) system of the liver. Inhalants, except nitrites, are depressants that act directly on the central nervous system through a wide range of mechanisms, which is yet to be completely elucidated.[31],[32] The intoxication lasts only a few minutes but can be extended for several hours by breathing inhalants repeatedly. The exact mechanism(s) responsible for tissue and organ damage with inhalant abuse have not yet been determined. Pulmonary uptake of a volatile substance depends upon a variety of factors including the concentration in inspired air, the air/blood and blood/tissue partition coefficients, ventilation, pulmonary blood flow, and the distribution of body fat.[33] As the chronic exposure continues distribution of inhaled compounds within the body generally follows a pattern of initial high concentrations in well perfused organs such as brain, liver, heart, and kidney followed by slow accumulation in tissues such as muscle and fat, with lesser blood supply.[23]

A few studies suggest that 25–40% toluene is exhaled unchanged via the lungs, and a greater proportion is metabolized and excreted via other pathways. The primary route of toluene metabolism is by hydroxylation to benzyl alcohol by members of the CYP family. It is believed that in humans, benzyl alcohol is metabolized to benzaldehyde by CYP rather than alcohol dehydrogenase.[34] Benzaldehyde in turn is metabolized to benzoic acid, primarily by mitochondrial aldehyde dehydrogenase (ALDH)-2 while only a small percentage is metabolized by cytosolic ALDH-1. Moreover, benzoic acid is metabolized to either benzoyl glucuronide or hippuric acid.[35] Benzoyl glucuronide is produced by the reaction of benzoic acid with glucuronic acid, which accounts for 10–20% of benzoic acid elimination. Hippuric acid is also known as benzoylglycine and is produced from benzoic acid in two steps:First benzoic acid is converted to benzoyl-coenzyme A (CoA) by the enzyme benzoyl-CoA synthase; then benzoyl-CoA is converted to hippuric acid by benzoyl-CoA: Glycine N-acyltransferase.[36] Hippuric acid is the primary urinary metabolite of toluene.

   The Role of Laboratory Top

Similar to the other substance abuse, appropriate medical care is very important for any inhalant user. In general, inhalants are not assessed along with routine urine drug screening. Hence detection relies on the clinical judgment of medical personnel who also administer screening questions as part of conducting a thorough history and physical examination. When considered in certain clinical contexts, abnormal nontoxicological laboratory results, such as elevated liver enzymes, can also indicate a suspicion of inhalant abuse. Some of the laboratory tests which are commonly been done for a patient presenting with acute inhalant intoxication or suspected inhalant use includes a complete blood count, determination of electrolyte, phosphorous, and calcium levels, an acid-base assessment, hepatic and renal profiles, and cardiac/muscle enzyme analysis. Specific urine drug testing is sometimes useful as part of the treatment-compliance plan when benzene, toluene, or a similar agent has been chronically abused, because major urinary metabolites (phenol and hippuric acid, respectively) are detectable when there has been a high level of use.[23] Detection and monitoring of these compounds often include analysis for the parent compound and its metabolites. Laboratory has a very important role in the substance abuse testing program.

   Analytical Issues Top

Collection and transportation

Proper sampling is a major issue in inhalant testing program. While collecting blood the chances of adulteration or dilution are very less. In a clinical setting, urine is a specimen of choice for inhalant testing, the validity of test depends on the integrity of the urine sample being collected from the patient. Adulterating or diluting the urine sample has been a common practice between the patients coming to a de-addiction clinic. It is therefore very important to take precautions while collecting a sample from the inhalant users. Sample should be collected in a clean, dry and leak proof container under close supervision. The sample's temperature (33°C–36°C) and pH (4.6–8) should be checked right after the collection. Any suspicion about the integrity of sample should be immediately informed to the laboratory staff.[37],[38]

Sample storage

Storage of urine sample is a major area of concern in inhalant testing. Since inhalants are not detectable in biological specimens for a longer period of time, it should be transported immediately after collection and always stored at −20°C till analysis. Urine samples have been found stable at 4°C for 5–6 days and at −20°C for longer duration (6 months).[39]

Biological specimen

Among all the biological samples, urine is the preferred biological specimen for toluene-based inhalants in a clinical setting. The significant advantage of using urine is that it is generally available in sufficient quantity and can be collected noninvasively. Drugs and their metabolites tend to be present in relatively high concentrations and remain detectable in urine from several hours to several days after the last use, especially in chronic users. The concentration of a drug in urine depends on a number of factors. In general, drug concentrations in urine vary with dose, route of administration, time elapsed since administration, individual's physiological status, which influences urine flow, urine pH, and metabolism. It also depends upon the metabolism and mode of elimination of the particular drug in the body. Moreover, urine has a simpler matrix than other biological specimens, which simplifies its sample preparation and analysis. Before carrying out a urine test, it is essential to ensure that urine specimen being tested has actually from the patient who is being assessed, as urine specimens can easily be tempered by substitution, dilution or adulteration.[37]

   Methodology for Detection Top

Preparation of biological samples for analysis

Biological samples are very complex multi-component mixtures. Often the analyte (compound) of interest is present in very low concentrations in the biological matrix. Sample preparation is, therefore, necessary to concentrate the analyte and remove the extraneous material that may interfere in the assay. The chief physiological methods used to isolate and enrich analytes include dilution, protein precipitation, hydrolysis, liquid-liquid extraction and liquid-solid extraction.[37],[38],[40]

Analytical techniques

The specific method chosen by a laboratory depends upon many factors that include nature and quantity of specimen, sensitivity required, existing facilities, workload, available man power and economic considerations etc., In general, drug abuse testing is a two tier process-preliminary screening, followed by confirmatory analysis of the prescriptive positive and doubtful results. Analytical methods used in most laboratories for the detection of drugs of abuse are so selected as to meet the requirements for screening and confirmation. A screening test should be able to identify potential positives and should be sensitive, rapid, and inexpensive, whereas the confirmatory tests should be more specific than screening test. Screening test generally involves color tests, immune-chromatographic test, spectrophotometric or colorimetric assays, immunoassays and thin layer chromatography.[38] More specific confirmation tests used to identify drugs of abuse are chromatographic techniques like headspace-gas chromatography (GC), gas liquid chromatography (GLC), high-pressure liquid chromatography (HPLC) and GC-mass spectrometry (MS), etc. The purpose of confirmation is to eliminate any false-positive results that may have originated from an initial screening process.

Several methods have been reported in literature for the determination of hippuric acid in urine based on color tests,[41],[42] immuno-chromatographic test,[43] spectrophotometric or colorimetric assays [44] and immunoassays.[45] More specific confirmation tests used to identify inhalant abuse are chromatographic techniques like headspace-GC, GLC,[23],[46],[47],[48],[49] HPLC,[50],[51] and GC-MS etc.[52],[53],[54]

Detection period

Detection period of inhalants depends on several factors. When interpreting the duration for the presence of inhalants abuse in the body, one must take into consideration variables including the body's metabolism, the subject's physical condition, overall body fluid balance, state of hydration, amount and frequency of inhalant usage, body mass, age, drug tolerance, and urine pH. It is necessary to understand the pharmacokinetic parameters of the drug of interest when interpreting data. Moreover, the detection of the inhalant use may also depend on the type of technique adopted by the laboratory. As stability of urinary metabolite of toluene, hippuric acid has been a matter of concern, use of filter paper to store the urine sample in dried form (dried urine spots) has also been reported in a recent study.[55]

Drug standards

Similar to other drugs of abuse, reference drug standards are also essential for testing inhalants in body fluids.[38] These reference standards are used in all determinations that require comparison to a chemical substance. No exact identification of a particular inhalant is possible without such standard. The number of inhalants to be screened in the laboratory will however depend upon the availability of reference drug standards. For example, Laboratory would require standards of hippuric acid and o-cresol for detection of toluene-based inhalants in urine or blood.

Cut-off values (toluene)

The problem with this diagnostic test is the lack of specificity of hippuric acid in the determination of toluene intoxication, as this acid is an endogenous component, deriving from the metabolism of diet components. Urinary concentrations of up to 1.5–1.6 g hippuric acid per g of creatinine are considered normal. When higher levels are detected, exposure to toluene is assumed. Exposure is considered excessive when hippuric acid levels in the urine are higher than 2.5 g per g of creatinine.[7],[23],[56]

   Interpretation of Laboratory Results Top

Communication with the laboratory

Regular interaction of clinical staff with the laboratory personnel is necessary so that lacunae on either side are minimized in the interest of patient care. Clinicians should be familiar with the strength and limitation of substance abuse testing programs. For meaningful interpretation of laboratory results, particulars of inhalant use are very important. Hence, clinicians should furnish adequate clinical history regarding type of inhalant used, mode of administration, last dose, frequency of use, quantity of consumption and medicines being prescribed to the patient. Such information will be helpful for meaningful interpretation of the test results.[38],[39]


The diagnostic test of toluene abuse has certain limitation too. Hippuric acid can be found in both inhalant users as well as non-users/healthy subjects as it is a metabolite of normal diet. The problem with this diagnostic test is the lack of specificity of hippuric acid in the determination of toluene intoxication, as this acid is an endogenous component, deriving from the metabolism of diet components.[7] For instance, urinary hippuric acid may be due to ingestion of benzoate preservatives in foods and drinks and not exposure to toluene, raising the possibility of false-positive findings. This is one of the major confounder in hippuric acid evaluation.[7],[57] Therefore, the ratio of urine hippuric acid level to urine creatinine level should be taken into consideration to detect toluene abuse. Thus, caution must be used when interpreting the laboratory result.

   Conclusion Top

Efforts are under way to improve laboratory diagnosis of inhalant use and abuse,[7],[58] but such tests are not yet widely available, nor have they been implemented in routine clinical practice. Findings from the occupational toxicology and inhalant abuse literature suggest that bioassays for hippuric acid, o-cresol levels, and benzylmercapturic acid may eventually be useful urinary markers of toluene abuse.[23],[58],[59],[60],[61] Keeping in view that toluene is primarily excreted in the urine as the metabolite, hippuric acid and excretion of hippuric acid is usually complete within 24 h after exposure. Screening of urinary hippuric acid could aid the diagnosis of recent and chronic toluene-based inhalant abuse in a clinical setting. Regular lab screening for inhalant abuse as well as other substance abuse and health risk behaviors must be a part of standard clinical care.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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