Pre-analytical stability of drugs of abuse in urine for confirmatory testing: a systematic review.

Abstract

Assessment of drugs of abuse in biological fluids requires thorough knowledge of stability of the drugs under various conditions, including sample collection, handling, transportation, and analysis, to ensure accurate interpretation of results. This systematic review provides an overview of the literature on the pre-analytical stability of selected clinically relevant drugs of abuse in urine. A systematic search of the PubMed and Embase databases was conducted in October 2020 and February 2024. The search strategy encompassed over 20 drugs and their relevant metabolites tested in urine, focusing on studies that examined the stability of opioids, amphetamine-like drugs (including ephedrine, cocaine and cathinone), and cannabis using mass spectrometry. A total of 2688 records were identified, and 71 studies met the inclusion criteria. These studies evaluated storage conditions including room temperature, refrigeration, freezing, and deep freezing, as well as the effects of freeze-thaw cycles. Most drugs demonstrated stability for months when refrigerated or frozen, and deep freezing and freeze-thaw cycles generally had minimal impact on stability. However, storage at room temperature showed limited stability, with cathinone, cannabis, morphine, codeine, and cocaine being particularly prone to degradation under different conditions. This review offers valuable insights into the storage stability of a wide range of drugs of abuse in urine, serving as a practical resource for healthcare professionals and others working with these substances in laboratory settings.

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Collection date 2026 Mar.

Author notes

Introduction

Confirmatory testing for drugs of abuse is, for example, routinely performed in laboratories for purposes such as clinical evaluation, workplace drug testing, or compliance supervision. Both positive and negative test results can have significant consequences for the individual being tested. Knowledge of the stability of drugs of abuse in testing material, often urine, is essential for accurate interpretation of results. A lack of information on storage and transport conditions can lead to incorrect conclusions or evaluations, affect interpretation, and potentially result in unjustified legal consequences [1, 2].

Urine is a matrix that is easy to sample by a non-invasive technique but is also relatively easy to manipulate if not obtained under supervision. Since urine may contain both the parent drug and its metabolites, the design and the analytical method should encompass both conjugated and free metabolites produced through biotransformation. Detection of drug metabolites provides valuable information for clinicians. Their presence indicates the metabolism of the parent compound and can help rule out whether drugs have been added directly to the sample in an attempt to manipulate it. Additionally, detecting metabolites can extend the window of detection and potentially reduce false-negative results. For example, French et al. reported that parent compounds, such as morphine, are eliminated significantly more rapidly than their glucuronide metabolites [3]. Further, certain drug concentrations may be higher in urine compared to other matrices. The drug may change structure by degradation or exposure to different environments [4].

Urine drug testing can include an initial screening using an immunological assay, followed by a confirmatory test for positive test results. Confirmatory analysis of drugs of abuse in urine is conducted using gas chromatography–mass spectrometry (GC–MS), liquid chromatography-mass spectrometry linked to quadrupole time of flight detection (LC–MS–QTOF), liquid chromatography-tandem mass spectrometry (LC–MS/MS), or by liquid chromatography–mass spectrometry (LC–MS). These methods provide sensitive and specific techniques for confirmatory drug analysis [1].

The time span between sample collection and laboratory analysis is typically several days, making knowledge on the pre-analytical stability of the drugs in urine highly important. Furthermore, repeated testing — such as for analytical purposes, forensic testing, or inconclusive or questionable results — may further extend the relevant time frame.

Guidelines for drugs of abuse testing touch only briefly on the stability of common drugs of abuse and their metabolites [5, 6]. The third edition of the European Guidelines for Workplace Drug Testing in Urine recommends that samples be “refrigerated” for short-term storage and “frozen or below” for long-term storage without defining specific temperature intervals [1]. These guidelines provide useful directions for drug testing in general but have sparse directions for storage based on the instability of some drugs.

Several studies have investigated drug stability in urine; however, the findings were limited [4].

Conducting a systematic review, the study aimed to gather knowledge on the pre-analytical stability of drugs of abuse in order to summarize the available stability data to provide a helpful tool to, for example, healthcare professionals and other professional groups working with and interpreting confirmatory drug testing.

Materials and Methods

Data sources and search strategy

The present systematic review adheres to the PRISMA (Preferred Reporting Items for Systematic

Reviews and Meta-Analyses) statement [7]. The protocol was registered on PROSPERO [8]. Databases searched included Embase (all years) and PubMed (all years) with no filters.

PubMed and Embase were searched on October 23, 2020, and February 28, 2024. Search strings are shown in the Supplementary Table S1 (see online supplementary material for this table).

The following drugs were included: morphine, heroin, codeine, buprenorphine and its metabolite norbuprenorphine, methadone and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), tramadol and its metabolite o-desmethyltramadol, cocaine and its metabolite benzoylecgonine, amphetamine, methamphetamine, 3,4 methylenedioxyamphetamine (MDA), 3,4 methylenedioxymethamphetamine (MDMA), methyldiethanolamine (MDEA), ephedrine, cathinone, and tetrahydrocannabinol (THC) and its metabolite 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH). To identify studies investigating stability, the following search terms were used: validation, storage, and stability, and to restrict on the matrix, the search term of choice was: urine. Keywords, free text words, Emtree headings and MESH terms were included in the search strings (Supplementary Table S1, see online supplementary material for this table).

A screening strategy based on three steps was implemented to identify relevant articles using Covidence systematic review software, Veritas Health Innovation, Melbourne, Australia, available at http://www.covidence.org (Fig. 1). Titles and abstracts were screened independently and blinded to each other’s selection. Then, the full text was read, and consensus was reached on the final list of included studies. The screening process is described in Fig. 1.

Figure 1.Flow diagram of the inclusion and exclusion procedures. PRISMA, Preferred Reporting Items for Systematic Meta-Analysis; GC–MS: gas chromatography–mass spectrometry; LC-MS-TOF: liquid chromatography-mass spectrometry linked to quadrupole time of flight detection; LC-MSMS: liquid chromatography-tandem mass spectrometry; HPLC-MS: high pressure liquid chromatography-mass spectrometry.

Data extraction

Extracted data included author details, year of publication, journal, investigated drugs and metabolites, method of analysis, tested concentrations, temperature, duration of storage, stability criteria, and data presentation.

Due to the use of varying temperature classifications across the publications, the following standardized temperature categories were introduced by the authors to facilitate comparison between studies: room temperature (20–25°C), refrigerated (4–8°C), frozen (−20 to −22°C), deep frozen (−60 to −80°C). If a study referred only to a general term such as ‘room temperature’ without specifying a range, it was recorded as ‘room temperature (20–25°C)’. Studies that did not indicate specific temperature ranges are noted in Tables 1–3 and Supplementary Table S2 (see online supplementary material for a colour version of this table). The number of freeze–thaw cycles was also recorded where reported.

Table 1.Published data on stability of amphetamine-like drugs in urine

Drug	Storage condition
	Room Temperature (20–25°C)			Refrigerated (4–8°C)			Frozen (–20°C)			Deep Frozen (–80°C)	Freeze–thaw cycles
	8h–3d	2w	5–6m	1–3d	1–2w	3–6m	2w	57d–6m	12m	1m	3 times
AMP	8h [11, 12]; 1d [13–16]	[17]	5m [18]; 6 m [9, 19]	1d [20]	1w [13, 14]; 2w [16, 17]	3m [21]; 5 m [18]; 6 m [9, 19]	2w [11, 13]	57d [22]; 6 m [9, 12]	9m [21, 23]; 12 m [24]	[20]	[10–13, 15, 16, 21, 22, 25–27]
METAMP	8h [11, 12] ; 1d [13–16]; 3d [28]	[17]	5m [18]; 6 m [9, 19]	1d [20]	1w [13, 14]; 2w [16, 17]	5 m [18]; 6 m [9, 19]	2w [11, 13]	1m [28]; 57d [22]; 6 m [9, 12]	12m [24]	[20]	[10–13, 15, 16, 22, 25–28]
MDA	8h [11, 12] ; 14 h [29]; 1d [13–16]	[17]	6m [9]	1d [20] 3d [29]	1w [13, 14]; 2w [16, 17]	6m [9]	2w [11, 13]	57d [22]; 6 m [9, 12]		[20]	[10–13, 15, 16, 22, 25–27, 29]
MDMA	8h [11, 12] ; 14 h [29]; 1d [13–16]	[17]	6m [9]	1d [20] 3d [29]	1w [13, 14]; 2w [16, 17]	6m [9]	2w [11, 13]	57d [22]; 6 m [9, 12, 30, 31]		[20]	[10–13, 15, 16, 22, 25–27, 29–31]
MDEA	14h [29]; 1d [15, 16]	[17]	poor response [9]	1d [20] 3d [29]	2w [16, 17]	poor response [9]		6 m [9]		[20]	[15, 16, 26, 27, 29]
EPH	1d [16]; 3d [28]			1d [20] 3d [32]	2w [16]			1m [28]		[20]	[16, 26–28]
CAT	1d [9, 16]		n.d. [9]	1d [9, 20]	2w [16]	n.d. [9]		6 m [9]		[20]	[16]	[10]

All studies used spiked drug-free urine from volunteers, except the following studies, which used commercially available control material (e.g., BioRad Laboratories, UTAK, nal von minden GmbH, Hydrocarbons Inc.): Dahlin et al. [13], Paul et al. [10], and Wang et al. [22]. Dugan et al. [24]. Description of storage temperature matched only partly the classifications used in this review, as room temperature was only described as “room temperature” without a specific temperature interval. The following temperature classification was introduced by the authors: room temperature 20–25°C, refrigerated 4–8°C, frozen −20 to −22°C, deep frozen −60 to −80°C. The specific temperature intervals used to describe storage conditions were declared or differed for the following studies: Dahlin et al. room temperature RT (25 °C) [13]; Guo et al. RT (25 °C) [11]; Hughes et al. RT (25 °C) [19]; Zaitsu et al. RT (25 °C) [18]; Zhang et al. RT (18 °C) [16]. Patient samples were used in the following studies: Cherma et al. [21]; Del Mar Ramirez Fernandez et al. [26, 27]; Dugan et al. [24]; Guo et al. [11]; Malaca et al. [15]; Pichini et al. [30]; Stephanson et al. [28]. This table presents stability data for amphetamine like drugs, ephedrine and cathinone in urine. Green fields: all studies showed stability; orange fields: includes studies that show instability (degradation > 20%) or poor response (poor drug detection).

Table 2.Published data on stability of THC and THC-COOH in urine

Drug	Storage condition
	Room Temperature (20–25°C)		Refrigerated (4–8°C)			Frozen (–20°C)			Deep frozen (–80°C)	Freeze–thaw cycles
	1h–3d	7–8 d	1h–3d	7–21d	1–6m	3d–15d	2–6m	12m	12m	3 cycles
THC–COOH	1h [46]; 8 h [38]; 16 h [33]; 1d [42, 52]; 3d [58]	8d [50]	1h [46]; 2d [34]; 3d [33, 35, 38, 52]	7d [39, 50, 59]; 10d [51]; 21d [60]	1m [36]	3d [52]; 14d [34]	4 m [39]	[36]; [24]	[56]	[33, 35, 38, 52, 57, 61]^a
	21h [35]	8d [50]					4 m [39]
	21h [35]	7d [34, 36]					2 m [37]
THC	8h [38]; 1d [52]		3d [33, 35, 38, 52]		1m [55]; 78d [53]	3d [52]	2m [55]			[35, 38, 52, 55]^b
THC	16 h [33]; 21 h [35]		3d [33, 35, 38, 52]		1m [55]; 78d [53]	3d [52]	2m [55]			[33]
THC-glucuronide	16h [33]; 21 h [35]	7d [36]	3d [33, 35]		6 m [36]		6m [36]			[33, 35]
THCCOOH-glucuronide	16h [33]; 21 h [35]	7d [36]	3d [33, 35]	7d [39]; 10d [51]; 21d [60]	6m [36]	15d [34]	4 m [39]; 6 m [36]			[33, 35]
THCCOOH-glucuronide	2d [34]	7d [36]	3d [33, 35]	7d [39]; 10d [51]; 21d [60]	6m [36]	15d [34]	4 m [39]; 6 m [36]			[33, 35]

One study used spiked drug-free urine from volunteers with sodium azide [51]. All studies used spiked drug-free urine from volunteers, except the following studies, which used commercially available control material (e.g., BioRad Laboratories, UTAK, nal von minden GmbH, Hydrocarbons Inc.): Giardino et al. [53] and Stout et al. [46]. Description of storage temperature matched only partly the classifications used in this review, as room temperature was only described as “room temperature” without a specific temperature interval. The following temperature classification was introduced by the authors: room temperature 20–25°C, refrigerated 4–8°C, frozen –20 to –22°C, deep frozen –60 to –80°C. The specific temperature intervals used to describe storage conditions were declared or differed for the following studies: Giardino et al. R (2–8°C) [53]; Pellegrini et al. RT (25°C) [42]; Romberg et al. F (–15°C) [37]; Skopp et al. RT (20°C) [51]; Stout et al. RT (25°C); Wei et al. RT (25°C) [50]. Studies that did not define stability for the conjugated forms: Abraham et al. [52], Aizpurua-olaizola et al. [57], Dugan et al. [24], Frei et al. [58], Giardino et al. [53], Gonzales et al. [55], Müller et al. [56], Nestic et al. [59], Pellegrini et al. [42], Romberg et al. [37], Sempio et al. [38], Sharma et al. [61], Stout et al. [46] Wei et al. [50]. Patient samples were used in the following studies: Desrosiers et al. [36]; Dugan et al. [24]; Romberg et al. [37]; Sim et al. [60]; Skopp et al. [34]. This table shows the available stability data for cannabis (THC) and its metabolite (THC-COOH). Green fields: all studies showed stability; Orange fields: studies present data for instability. Patient samples [24, 34, 36, 37]; the others are spiked samples.

Table 3.Published data on stability of opioids and cocaine in urine

Drug	Storage condition
	Room Temperature (20–25°C)		Refrigerated (4–8°C)			Frozen (–20°C)			Deep Frozen (–80°C)	Freeze–Thaw cycles
	4h–7d	11m	1d–21d	1m	6m–1y	5d–2w	1m	57d–1y	5d–6w	3 cycles
BUP	4h [68]; 16 h [69, 70]; 1d [13, 71–73]		3d [69, 70]; 7d [13]; 21d [74]	[68]	6m [55]	5d [69]; 2w [13]	[68, 73]	57d [22]; 85d [71]; 6 m [55]; 1 y [68]		[13, 22, 55]^a; [64, 68–70, 72, 73]
NORBUP	4h [68]; 6 h [69, 70]; 16 h [70]; 1d [13, 71–73]		3d [69, 70]; 7d [13], 14d [74]	[68]	6m [55]	5d [69]; 2w [13]	[68, 73]	85d [71]; 6 m [55]		[13, 55]^a; [64, 68–70, 72, 73]
MET	1d [13, 41, 75]; 3d [76]; 7d [77]		1d [19, 41]; 7d [13]		6 m [55]	5d [41]; 2w [13]		57d [22]; 6 m [55]	5d [20]	[13, 41]^c; [22, 25, 55]^a; [64, 76, 77]^b
MOP	5h [43]; 7 h [62]; 16 h [70]; 1d [13, 41]	[40]	1d [41]; 3d [70]; 7d [13]		6m [55]; 1 y [45]	5d [41]; 2w [13]		57 d [22]; 6 m [55]; 1 y [24, 45, 63]	6w [43]	[13, 41]^c; [22, 25, 43, 55]^a; [70]; [64]; [62]
MOP	1d [42]	[40]	1d [41]; 3d [70]; 7d [13]		11m [40]	10d [62]		11m [40]; 2 y [63]	6w [43]	[13, 41]^c; [22, 25, 43, 55]^a; [70]; [64]; [62]
COD	16h [70]; 1d [13, 41]	[40]	1d [41]; 3d [70]; 7d [13]		6m [55]; 1 y [45]	5d [41]; 2w [13]		57d [22]; 6 m [55]; 1 y [24, 45]		[13, 41]^c; [22, 25, 55]^a; [70]; [64]
COD	16h [70]; 1d [13, 41]	[40]	1d [41]; 3d [70]; 7d [13]		11 m [40]	5d [41]; 2w [13]		11m [40]		[13, 41]^c; [22, 25, 55]^a; [70]; [64]
TRM	4h [78, 79]; 8 h [80]; 16 h [70]; 27 h [81]; 1d [13]		1d [78, 79]; 27 h [81]; 3d [70]; 7d [13]		6 m [55]	7d [80, 82]; 2w [13, 78]		57d [22]; 6 m [55]		[13, 22, 55]^a; [64, 70, 78–81]^c; [82]^b
COC	1d [13, 41, 65]		1d [20, 41]; 3d [83]; 7d [13]		6m [55]; 1 y [84]^d; 60d [84]^e	5d [41]; 2w [13]		57d [22]; 3 m [66]; 6 m [55, 67], 1 y [84]	5d [41]	[13, 41]^c; [22, 25, 55]^a; [64–66]^a
COC	1d [42]		1d [20, 41]; 3d [83]; 7d [13]		6m [55]; 1 y [84]^d; 60d [84]^e	5d [41]; 2w [13]		2m [37]; 1 y [24]	5d [41]	[13, 41]^c; [22, 25, 55]^a; [64–66]^a

All studies used spiked drug-free urine from volunteers, except the following studies, which used commercially available control material (e.g., BioRad Laboratories, UTAK, nal von minden GmbH, Hydrocarbons Inc.): Chang et al. [63], Dahlin et al. [13], del Mar Ramirez Fernandes et al. [64], and Wang et al. [22]. Description of storage temperature matched only partly the classifications used in this review, as room temperature was only described as “room temperature” without a specific temperature interval. The following temperature classification was introduced by the authors: room temperature 20–25°C, refrigerated 4–8°C, frozen −20 to −22°C, deep frozen −60 to −80°C. The specific temperature intervals used to describe storage conditions were declared or differed for the following studies: Dahlin et al. RT (25 °C) [13]; Kacinko et al. RT (22 °C) [69]; Lin et al. RT (25–30°C) and F (−15°C) [40]; Menotti et al. R (4–8°C) [83]; Pellegrini et al. RT (25 °C) [42]; Romberg et al. F (−15°C) [37]. Patient samples were used in the following studies: Del Mar Ramirez Fernandez et al. [64]; Dugan et al. [24]; Lin et al. [40]; Lin et al. [70]; Mariottini et al. [74]; Paul et al. [67]; Romberg et al. [37]; Rosado et al. [65]; Wang et al. [62]. The studies described the tested samples as “spiked” when blank urine was used, and as “fortified,” when calibration standards, stocks, or controls were used. This table presents the available stability data on opioids in urine. Green fields: studies showed stability; orange fields: studies present data for instability.

Drugs, methods of analysis, storage conditions, and the tested concentrations are summarized in Supplementary Table S2 (see online supplementary material for a colour version of this table).

The drugs were grouped into (i) amphetamine-like drugs; (ii) THC and THC-COOH; and (iii) opioids and cocaine for the following data mining. The category “amphetamine-like drugs” includes the following: amphetamine, methamphetamine, MDA, MDMA, MDEA, ephedrine, and cathinone.

Results

Study selection

The process of inclusion and exclusion is shown in Fig. 1. In total, 2688 articles were identified from PubMed and Embase. After screening titles and abstracts based on the inclusion criteria, 2617 articles were excluded (see Fig. 1 for inclusion and exclusion criteria). Finally, 71 studies were included. Some studies included full method validations (n = 55) while other studies exclusively focused on pre-analytical stability (n = 16). One study investigated pre-analytical stability in relation to retesting.

Study description

All studies were published between 1991 and 2024. Studies and the extracted data are summarized and described in Supplementary Table S2 (see online supplementary material for a colour version of this table). Pre-analytical stability has been investigated in various types of studies, including validation studies, clinical studies focusing on drug administration, and studies evaluating different sample tubes or the use of preservatives in their study design. Studies using preservatives in their design were only included in this review with respect to results without additives, since preservatives are rarely used for screening and testing outside of forensic settings. The tables include information on whether a study used commercially available control material with preservatives. Stability results from study designs based on preserved control material were comparable to results from studies using spiked drug-free urine. The preservatives used to stabilize the control material and protect the target compounds from degradation in the presence of bacteria did not appear to affect the stability results at the tested concentrations. Results were mainly presented as percentage bias from the investigated target concentration. Analysed concentration levels spanned several orders of magnitude.

Amphetamine-like drugs

The pre-analytical stability of amphetamine-like drugs in urine was investigated in 25 of the included studies (Table 1). Apart from three studies using patient samples, all studies used spiked urine. The confirmatory methods used in these studies was either GC–MS (n = 10), LC–MS/MS (n = 14), or LC–MS-QTOF (n = 1). The stability criteria, which were used in up to 62% of the studies, were: a non-significant change or a change < 20% compared to baseline. Different bias acceptance levels were applied, 10, 15, or 20%. Studies specifying results to draw conclusions on stability, most often reported a relative change in concentration compared to baseline values. A significant number of studies (46%) did not present quantitative results. The tested drug concentrations varied greatly among the studies.

The studies of amphetamine-like drugs investigated stability under several storage conditions (see Table 1). All amphetamine-like drugs were stable in urine at −80°C for one month and at least at −20°C for 6 months, as well as when subjected to at least three freeze–thaw cycles. One study showed poor analytical response for MDEA and cathinone after storage at room temperature, and after refrigeration for 6 months [9]. Another study showed degradation of cathinone after three freeze-thaw cycles [10]. Besides this, amphetamine-like drugs were stable under the other investigated conditions (room temperature for 6 months (apart from ephedrine and cathinone), refrigerated for 6 months (apart from ephedrine and cathinone), −20°C for 6 months (apart from ephedrine).

THC and THC-COOH

THC and THC-COOH were investigated in 22 of the included studies (Table 2). The confirmatory methods used in these studies were either GC-MS (n = 12) or LC-MS/MS (n = 10). These studies were divided between stability studies only (n = 12) and method validation studies (n = 10). For THC-COOH, the analysed concentrations ranged from 0.04 to 4000 ng/mL, and showed great variation in concentrations. Respectively, for THC the analysed concentrations spanned from 1.5 to 4000 ng/mL. The studies showed conflicting results for THC and THC-COOH, with some reporting stability and others observing degradation under comparable conditions. Both compounds were generally stable over three freeze-thaw cycles and during storage for one year at −80°C; only one study out of eight reported instability of THC after three freeze-thaw cycles [33]. For THC and THC-COOH, four studies showed instability at room temperature when stored at different periods of time (from eight hours to seven days) [33–36], and seven studies presented results that indicated the drug and its metabolite were stable at room temperature and not degraded (Table 2). Studies using authentic patient samples presented results where the analytes were unstable at room temperature after two to seven days [34], when refrigerated for more than one month [36], and when frozen for 2 months [37]. No evident association between the use of authentic or spiked urine samples and stability results was observed. Sempio et al. found that all analytes were stable, within ±20% of baseline concentrations, when stored refrigerated or frozen. To ensure accurate reanalysis of urine specimens for cannabinoids, storage at −20°C was recommended [38]. Felli et al. reported that THC-COOH and THC-COOH-glucuronide were stable for seven days at 4°C and for 120 days at −20°C in 10 authentic urine samples [39].

Opioids and cocaine

Opioids were investigated in 35 studies. The analytical method applied was GC–MS in 14 studies, and LC–MS or LC-MS/MS in the remaining 21 studies. Authentic patient samples were used in four studies, while spiked urine was the material of choice in the remaining 31 studies. A definition of the applied stability criteria was given in 15 studies (43%). A minor part of the studies (23%), reported no quantitative results. Baseline concentrations showed great variation. For morphine, for example, the concentrations ranged from 5 to 1000 ng/mL, and for cocaine from 25 to 1000 ng/mL.

Buprenorphine, norbuprenorphine, methadone and tramadol were generally stable under different conditions (Table 3). A study by Lin et al. using patient samples [40] showed that morphine and codeine partially degraded under three different conditions (11 months at room temperature, refrigerated, and frozen). Some researchers found morphine and cocaine to be stable at room temperature for 24 hours [13, 41], while Pellegrini et al. reported both compounds to be unstable under this condition [42]. Dugan et al. found that cocaine was not stable for 1 year at −20°C [24]. Xu et al. reported acceptable stability for three freeze-thaw cycles. Additionally, Xu et al. found that urine samples could be stored at −80°C for 6 months without morphine degradation [43]. Moody et al. [44] observed that morphine remained stable at -20°C for 466 days, with concentration decreases of less than 15%. Morphine was also stable independently of urine pH, with differences of less than 20%, not only when frozen, but also when refrigerated [45].

Discussion

This systematic review gathered knowledge on the pre-analytical stability of common drugs of abuse in urine at different storage conditions used in routine laboratory testing.

Correct storage of patient samples is one important part of the analytical process for ensuring reliable test results. Knowledge of parameters such as drug stability, drug administration form, consuming frequency, storage temperature, time, and urine preparation is important for interpreting analysis results.

The concentrations of drugs measured can be influenced by properties of the sample such as pH, which may be temperature dependent, by prolonged transport, and by adhesive properties of the container material, which have been shown to alter cannabis results [46, 47]. Metabolic degradation, chemical transformation, or a combination of both may lead to falsely low results. Further, elevated temperatures can lead to decreasing creatinine concentration in urine thus limiting its use as a validity parameter [47]. Knowledge of these factors and their influence on sample stability allows laboratories to react and adjust pre-analytical procedures to ensure correct drug concentration measures.

In this systematic review, stability results from study designs based on preserved control material were comparable to results from studies using spiked drug-free urine. The preservatives used to stabilize the control material and protect the target compounds from degradation in the presence of bacteria did not appear to affect the stability results under the applied concentrations.

Subsequently, the three analysed drug groups are discussed separately.

Amphetamine-like drugs

The amphetamine-like drug group shares pathways of metabolism and has similar effects in the body. Apart from cathinone, and to a lesser degree MDEA, more frequently analysed amphetamine-like drugs were stable at the reported conditions. For storage at room temperature and when refrigerated, Alsenedi et al. described an initial degradation of the drug cathinone with almost none left after 21 days. The authors concluded that urine must be frozen immediately after collection to prevent its degradation [9]. It has been shown that cathinone degradation of is pH dependent [48]. Validity testing for pH helps to detect adulteration attempts but can also reveal a cause of cathinone degradation. Methamphetamine in urine slightly contaminated with bacteria was previously demonstrated to be stable for 150 days when stored at 25°C without the addition of any preservative [18].

In summary, most studies agreed that amphetamine-like drugs (amphetamine, methamphetamine, ecstasy, and ephedrine) were stable under all analysed conditions (room temperature and refrigerated up to 6 months, at −20°C up to 12 months, −80°C up to 1 month, and after three freeze-thaw cycles). Cathinone was stable when the drug was stored at −20°C with concentrations within ±20% of the original spiked concentration [9, 20], but the drug degraded at room temperature.

THC and THC-COOH

Cannabis is the most commonly used drug worldwide with estimated 209 million people in 2020 using cannabis, representing 4% of the global population [49]. Although THC-COOH is the metabolite of primary interest, data regarding other metabolites are included to present a complete report of the available data.

Cannabinoid instability in urine presents a significant challenge in the laboratory setting. Urine samples suspected of containing cannabinoids should be handled with care in accordance with strict laboratory guidelines. Several factors—including oxidative degradation, microbial growth (fungi and bacteria), and storage conditions (e.g. tube selection, pH, and temperature) [33, 36, 46, 50]—can affect cannabinoid concentrations in urine and potentially lead to decreased concentrations.

THC-COOH was stable for 16 hours at room temperature and after three freeze-thaw cycles (Table 2). Glucuronide THC-COOH conjugates were unstable, undergoing hydrolysis to the unconjugated form during storage [51]. Storage-induced hydrolysis may affect the measured concentrations, as may be the case in the study by Scheidweiler et al. showing degradation at room temperature [33]. Different hydrolysis procedures, e.g. enzymatic, alkaline, or a combination of both, result in differing analyte recoveries and could explain variation in reported cannabinoid stability [35, 52].

Desrosiers et al. proposed that degradation in authentic patient samples could be explained by high intersubject variability due to differences in urinary pH, as higher pH leads to a decrease in the glucuronide forms and increased THC-COOH concentrations [34, 36, 51]. This could potentially influence the concentrations measured if the method used only targeted either the free or the conjugated forms. Results obtained from spiked urine suggested that acidification to pH 5 may help to enhance the stability of the analytes, but the liberation of THC-COOH from its glucuronide cannot be completely prevented [34].

Two studies described a loss of urinary THC-COOH, which could be due to its adhesiveness to sample test tube plastic during storage [53, 54]. Fraga et al. observed a 22% decrease in concentration at room temperature for ten days and an 8% decrease in refrigerated samples stored in polypropylene tubes for 4 weeks [54]. In addition, Giardino et al. reported insignificant changes in THC-COOH concentration when stored in polypropylene at 2–8°C for up to 42 days [53]. Both the polypropylene and polyethylene bottle systems tested resulted in small losses of THC-COOH from urine specimens [46]. Additionally, THC-COOH loss appeared to stabilize rapidly, and Giardino et al. concluded that no further loss from urine was observed over a week’s storage time [53]. Storage of biological specimens in glass tubes presents safety concerns for routine use personnel and is usually avoided.

It has been demonstrated that THC-COOH concentrations decline over time even when stored frozen [37, 55]. These authors proposed that the loss of this analyte could be due to the decreased solubility of the compound and its adherence to the sample tube walls. However, several studies confirmed that samples can be stored at −80°C for one year and showed good stability in freeze–thaw studies [33, 35, 38, 52, 55–57].

Authentic urine samples were used in five studies for evaluating THC and THC-COOH- related stability [24, 34, 36, 37, 39]. The studies reported good stability of THC-COOH for 2 weeks at −20°C but described a loss of THC-COOH (20 to 24%) in 12 of 38 specimens after 5 days at room temperature and nearly 40% after 10 days at room temperature [34]. However, it should be emphasized that results obtained from authentic samples are very complex to interpret due to the broad range of pH values present in urine specimens [24, 37]. The instability at room temperature could be at least partly due to the broad range of pH values present in urine specimens.

In summary, frozen storage provided acceptable pre-analytical stability of THC-COOH in urine for up to 12 months (−20 and −80°C) [24, 36, 56]. THC-COOH showed acceptable stability for up to three freeze–thaw cycles. To overcome pre-analytical problems in practice, it can be favourable to cool the samples during transportation [51]. For storage at room temperature, there was almost consensus that samples were stable for one day, but when a longer storage period was needed, refrigerated conditions were favourable (Table 2).

Opioids and cocaine

The studies included in this review found that buprenorphine and norbuprenorphine were stable at least 1 day at room temperature, 6 months refrigerated and frozen; and for three freeze and thaw cycles (Table 3). Methadone and its metabolite EDDP were stable under all tested conditions (room temperature, refrigerated, −20°C, −80°C, and freeze–thaw cycles). Codeine was described as a very stable compound, even under refrigerated and at room temperature conditions. The stability was independent of storage temperature and pH in urine specimens [45]. An exception and the only source showing instability for codeine was the study by Lin et al., in which the authors observed instability of codeine in authentic patient samples after 11 months of storage under all tested conditions: at room temperature, refrigerated, and frozen [40].

Several authors agreed that freeze–thaw cycles did not affect morphine stability. Conclusions regarding the stability of morphine stored at −20°C, refrigerated, and stored at room temperature conditions, were contradictory. Some authors found acceptable stability of morphine for one day at room temperature and up to one year in the refrigerator and freezer at −20°C [13, 24, 41, 45]. Other authors, on the contrary, reported significant losses, which were presumably due to unfavourable pH conditions or bacteria growth in authentic samples: This included instability after 11 months at room temperature and refrigerated or 2 years frozen [40, 62, 63]. Pellegrini et al. observed a statistically significant decrease in morphine concentration starting from 1 day after collection [42].

For cocaine, most studies showed overall good stability (e.g. 1 day at room temperature; 7 days refrigerated, 1 year at −20°C) [13, 20, 22, 25, 41, 55, 64–67], while, in contrast, three studies showed instability at room temperature and at −20°C [24, 37, 42]. Pellegrini et al. observed a decrease in cocaine concentration starting from day one after sample collection [42]. All authors concluded that tramadol was stable in human urine under the investigated conditions (Table 3).

In summary, some studies reported conflicting results, with some showing stability and others showing instability under similar conditions (Table 3). Yet overall, published results suggest acceptable stability regarding the needs for clinical routine setting.

Strengths and limitations

The strength of this study is its detailed compendium of stability results for common drugs in human urine analysed by confirmatory methods for diagnostic testing purposes. The summary is presented in table form and allows an overview for pre-analytical decision-making in relation to the best storage condition. However, there are also limitations to consider. Studies analysing authentic patient samples showed more variation in concentrations and stability compared to studies performed with spiked samples. The studies generally showed large variations regarding study design, stability criteria, and analysed drug concentrations (Supplementary Table S2, see online supplementary material for a colour version of this table). The extraction of data from papers regarding cannabis was limited due to results being only shown graphically or reported in text.

Conclusion

The studies showed that most drugs remained stable in urine samples over a period of months when stored refrigerated or frozen. Storage at room temperature showed a reduced drug-specific stability window, which should be considered. Deep freeze conditions and freeze–thaw cycles had limited effect on stability. Special attention should be given to cathinone, cannabis, morphine, codeine, and cocaine as they were sensitive to degradation under certain conditions [9]. More studies are needed to fully understand the stability of drugs of abuse in urine under different storage conditions influenced by pH changes or bacteria contamination.

Supplementary Material

Contributor Information

Elke Hoffmann-Lücke, Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus N, 8200, Denmark; Department of Clinical Medicine, Health, Aarhus University, Aarhus, 8000, Denmark.

Ellen Hollands Steffensen, Department of Clinical Medicine, Health, Aarhus University, Aarhus, 8000, Denmark.

Mie Samson, Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus N, 8200, Denmark; Department of Clinical Medicine, Health, Aarhus University, Aarhus, 8000, Denmark.

Eva Greibe, Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus N, 8200, Denmark; Department of Clinical Medicine, Health, Aarhus University, Aarhus, 8000, Denmark.

Author contributions

Elke Hoffmann-Lücke (Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing—original draft), Ellen Hollands Steffensen (Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing—review & editing), Mie Samson (Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing—review & editing), and Eva Greibe (Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing—review & editing)

Supplementary data

Supplementary data are available at Journal of Analytical Toxicology online.

Conflict of interest

None declared.

Funding

This work was supported by the Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark (to E.G.).

Data availability

All data are incorporated into the article and its online supplementary material.

References

Untitled section

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Study description

Amphetamine-like drugs

THC and THC-COOH

Opioids and cocaine

Discussion

Amphetamine-like drugs

THC and THC-COOH

Opioids and cocaine

Strengths and limitations

Conclusion

Supplementary Material

Contributor Information

Author contributions

Supplementary data

Conflict of interest

Funding

Data availability

References

Untitled section

References

Associated Data

Supplementary Materials

Data Availability Statement

Additional materials