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Volume 14 No. 04
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Case Reports

Segmental Hair Testing of Triazolam to Unmask a Suspected Case of Idiopathic Recurrent Stupor

Emanuela Postiglione, MD1; Elena Antelmi, MD, PhD1,2; Monica Moresco, MSc1; Simona Pichini, PhD3; Fabio Pizza, MD, PhD1,2; Giuseppe Plazzi, MD, PhD1,2; Francesco Paolo Busardò, MD, PhD4,5
1Department of Biomedical and Neuromotor Sciences (DIBINEM), University of Bologna, Bologna, Italy; 2IRCCS Istituto delle Scienze Neurologiche, AUSL di Bologna, Bologna, Italy; 3Drug Abuse and Doping Unit, Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Rome, Italy; 4National Centre on Addiction and Doping, Istituto Superiore di Sanità, Rome, Italy; 5Unit of Forensic Toxicology (UoFT), Department of Anatomical, Histological, Forensic and Orthopedic Sciences, Sapienza University of Rome, Rome, Italy


Stupor is a diagnostic challenge at emergency department. Differential diagnosis includes idiopathic recurrent stupor, formerly attributed to “endozepine-4” accumulation. This condition has been recently questioned because many suspected cases resulted in exogenous benzodiazepine intake that eludes the conventional toxicological assay. In case of unexplained recurrent stupor, to extend the benzodiazepine search in nonconventional matrices can allow unmasking of hidden toxic behavior.


Postiglione E, Antelmi E, Moresco M, Pichini S, Pizza F, Plazzi G, Busardò FP. Segmental hair testing of triazolam to unmask a suspected case of idiopathic recurrent stupor. J Clin Sleep Med. 2018;14(4):697–699.


Stupor is a diagnostic challenge in emergency departments. Differential diagnosis covers epilepsy, recurrent hypersomnias, metabolic causes, structural brain lesions, intoxication, and idiopathic recurrent stupor (IRS)1. IRS was first pointed out at the beginning of the 1990s, when several cases, originating from different ethnicities and settings, of inexplicable recurrent stupor were consecutively reported.1,2 The characteristic rapid-fast activity on electroencephalogram (EEG), and the transient improvement of the vigilance level with a concomitant normalization of background activity subsequent to intravenous injection of Flumazenil, a benzodiazepine (BDZ) antagonist, were all suggestive of BDZ intoxication. However, at that time synthetic BDZ contamination was excluded by mass spectrometry analysis, whereas high levels of endogenous ligand of BDZ recognition site of GABA-A receptor “endozepine 4” was detected by means of high-performance liquid chromatography measurement, in serum and cerebrospinal fluid samples during the ictal episodes. Endozepine 4 was therefore proposed to be pathognomonic and likely pathogenic for IRS.3,4 This hypothesis has been questioned, and in fact starting from 1998 onward several reported cases were found to be linked to exogenous benzodiazepine intoxication that eluded the conventional toxicological assays.5,6 This occurrence was likely due to the poor sensitivity of some routine BDZ screening tests and the short panel of detectable molecules.7 In addition, the short half-life of most of these compounds does not allow retrospective objective confirmation of a suspected BDZ intoxication by testing in conventional biological fluids.


A 42-year-old man with a history of three episodes of stupor, lasting approximately 24 hours each, and recurrent over a period of 2 months came to our attention. Other than the episodes, the patient was completely healthy. The first episode occurred after his spontaneous awakening in the early morning. The patient was found to be unconscious, but responsive to painful stimuli. He was transported at the hospital, where he proved to be hemodynamically stable. Metabolic screening, brain computed tomography, and urinary screening for principal drugs of abuse and BDZs were all negative (cutoff for principal drugs of abuse 100 ng/mL, for BDZ 300 ng/mL), and EEG performed during the episode showed “diffuse anterior prominent rapid fast activity and right hemisphere theta activity.” During the subsequent 24 hours, the subject appeared drowsy, confused, disoriented, and lightly disinhibited with slurred speech and mental and motor slowing. He was completely amnesic of the episode. Progressively and spontaneously, he regained alertness and orientation, as well as some memories of the prior days. He was discharged with carbamazepine treatment up to 800 mg, and a diagnosis of “possible epilepsy.” After 1 month, a second episode occurred. At this time, he was found at home unconscious, lying on the floor. He was hospitalized in a stuporous state and again, the neuroradiological and biochemical analyses (including BDZ urine screening) were negative. Again, the subject progressively and completely recovered in 24 hours. The third episode occurred after 15 days at home, in the morning, when he was found unconscious at the desk in front of a computer. The patient was admitted to the hospital and all clinical and toxicological investigations were negative. A clinical course overlapping with the previous stupor attacks confirmed a diagnosis of IRS, and antiepileptic drugs were tapered up to withdrawal. When the patient underwent further diagnostic investigations, metabolic screening, brain MRI, and EEG were normal and toxicological screening was again negative. Sleep studies (48 hours continuous polysomnography, Multiple Sleep Latency Test) disclosed mild sleep apnea and borderline daytime sleep propensity. Cerebrospinal fluid routine analyses and hypocretin-1 assay were normal. Suspecting BDZ intoxication, hair testing for principal drugs of abuse and BDZ was carried out in house, revalidating a previously ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) developed assay.8 This methodology, based on substance detection on segmental hair analysis through its molecular weight and ion fragments formed by the same substance inside the mass spectrometer detector, has been used to disclose even a past single drug administration. Specifically, it has been applied to assess the deposition of diazepam and its metabolites in hair following a single dose of diazepam.9 Because this technique is a confirmatory analysis, there is no evidence of false-positive results for hair testing of BZD or other drugs of abuse. The limits of quantification for the different drugs of abuse ranged from 10 to 50 pg/mg hair. Specifically, that of triazolam was 10 pg/mg hair. A hair sample of 3.5 cm in length (corresponding to the previous 3.5 months) was collected from the posterior vertex region of the head, close to the scalp and then stored at ambient temperature until analysis. The hair shaft was decontaminated twice using 5 mL methylene chloride for 2 minutes, dried, divided into 5-mm segments from the origin close to the root to the distal end. About 20 mg of each hair segment were extracted by solid-phase extraction and 10 μL aliquot of the extract was injected onto the UHPLC-MS/MS system. This technique segmental hair analysis disclosed the sole presence of triazolam in the sixth and seventh segment at the concentration of 18.4 and 12.3 pg/mg, respectively (Figure 1), corresponded roughly to the previous 3 to 3.5 months, the time span when the last two of the three episodes of stupor occurred. Unfortunately, the first episode that dated back to 4 months prior could not be confirmed because the segment corresponding to that time frame was not available in the shaft due to the patient's hair being cut. The patient was informed of the hair analysis result; he continued denying BDZ intake but at follow-up of 18 months he was attack free.

Triazolam concentration in subsequent hair segments.

Concentration of triazolam from different 0.5-cm hair segments of a subject with a suspected case of idiopathic recurrent stupor. Each hair segment corresponds to the previous 15-day time window dating back to 3.5 months period. Segment 1 is closest to the scalp and reflects the most recent 15 days; segment 7 is furthest from the scalp and reflects the final 15 days of the 3.5-month period.


Figure 1

Triazolam concentration in subsequent hair segments.

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Given the scientific controversies on the real existence of IRS and, above all, considering the important medico-legal implications of fraudulent BZD intoxications (ie, Munchausen syndrome or Munchausen by proxy),7 this case shows the importance of objective assessment of hidden toxic behavior and sheds light on the importance of testing of nonconventional biological matrices as a tool to reach clinical diagnosis in cases of unexplained recurrent stupor/hypersomnolence.


Work for this study was performed at the Department of Biomedical and Neuromotor Science, University of Bologna, Bologna, Italy. All authors have obtained permission to publish from their institutions, have seen and approved the final version of the article, and took the responsibility for the conduct of the research. The authors report no conflicts of interest.







idiopathic recurrent stupor


ultra-high-performance liquid chromatography tandem mass spectrometry



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