Development of Liquid Chromatography–Tandem Mass Spectrometry Analytical Methods for the Study of Whole-Body,Head,and Trunk Uptake and Elimination of Methamphetamine in 5-Day Postfertilization Zebrafish Larvae Using the Quick,Easy,Cheap,Effective,Rugged,and Safe Technique

Abstract

Synthetic cathinones are drugs of abuse substituted for amphetamine-like stimulant drugs such as methamphetamine. In this study, methamphetamine was studied as a prototypical amphetamine-like drug as a first step toward establishing methods to study this entire drug class. The internal concentration of methamphetamine in zebrafish larvae was determined using matrix-matched calibration along with extraction and purification of samples using the quick, easy, cheap, effective, rugged, and safe technique in liquid chromatography–tandem mass spectrometry. Whole-body and head/trunk uptake and elimination in 5-day postfertilization zebrafish larvae were determined. A gradient method was developed using 5 mM ammonium formate with 0.1% formic acid and methanol with 0.1% formic acid as mobile phases, 10 min of total run time, and a 0.3 mL/min flow rate. The limit of quantification was 60 ng/mL, linearity with r² = 0.9991, and recovery values from 92% to 120%. The internal concentration of methamphetamine was quantifiable in whole-body homogenates within 15 min of uptake analysis. The internal concentration increased with time, whereas a biphasic elimination pattern was shown. With increasing length of exposure, a higher accumulation of drugs was found in the head than in the trunk.

Introduction

New psychoactive substances (NPS) are substances of abuse, either in pure form or a preparation that are not controlled under the Single Convention on Narcotic Drugs of 1961 or the 1971 Convention but may pose a public health threat.¹ As such substances emerge in illicit use, their properties are usually unknown, including addiction liability and potential for adverse events. As these drugs come under regulatory scrutiny, they are usually classified by various regulatory authorities, restricting their use, sale, and distribution. However, often this is done in a context of limited available information.

Synthetic cathinones (still often referred to by the colloquial name “bath salts”), United Nations Office on Drugs and Crime (UNODC),^2,3 are β-keto phenethylamine derivatives that are⁴ structurally similar to amphetamine, methamphetamine, and 3,4-methylenedioxy-methamphetamine and share stimulant effects.⁵ They are synthesized by illicit drug suppliers, modifying the chemical structure of cathinone (the β-ketone analog of amphetamine) to obtain new analogs aiming to circumvent the law.⁶ This diversity in chemical modifications has led to a large group of cathinone analogs with uncharacterized pharmacokinetics profiles.⁷ According to UNODC, over 160 synthetic cathinones were reported as of 2019. Similarly, among the 15 stimulants reported for the first time in 2021, synthetic cathinones were the largest group, with six new substances. In addition, some modifications were novel for this group, like a halogen and a methyl group substitution in the phenyl ring.⁴

The number of new synthetic cathinones is increasing. Based on clinical case reports, the health problems, and psychological side-effects, linked with synthetic cathinones include depression, panic attacks, tachycardia, seizures, multiple organ failure, paranoia, psychosis, and death.^2,8–10 However, human and animal studies on these new synthetic cathinones are very limited. Therefore, information on pharmacological and toxicological properties still needs to be discovered.

Most studies involve rodents for pharmacological and toxicological evaluation. At the rate at which these new analogs of synthetic cathinones emerge, high-throughput approaches are needed. Based on the substantial cost and time investment that rodent studies demand, there is a need for a translational animal model with high-throughput capabilities. Larval zebrafish (i.e., 5-day postfertilization [5-dpf]) can serve as an alternative animal model that can be used for high-throughput drug screening. Zebrafish have conserved pharmacological responses based on high genomic and proteomic homology and nervous system structures comparable to humans. As early as 5-dpf, zebrafish express all major brain structures, neurotransmitters, and receptors.¹¹

No studies have been done on the toxicokinetics of synthetic cathinones in zebrafish larvae. Ignoring this critical issue can lead to poor outcomes and unreliable interpretation of observed effects in pharmacological studies. Thus, the evaluation of the internal concentrations of cathinones in larval zebrafish by chromatographic methods will contribute to a better understanding of the toxicokinetic properties of synthetic cathinones. The increasing number of NPS, particularly synthetic cathinones, makes the development of such methods more important.

As a starting point for addressing the various analytical challenges associated with studying larval zebrafish, the prototypical stimulant drug methamphetamine was used in the present studies, to address the need for reliable and reproducible analytical methods to detect these types of drugs. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) has been reported to effectively determine concentration of small molecules, pesticides, and some opioids in zebrafish.^12–16 Other associated challenges are the sample extraction and clean-up methods before LC-MS/MS, and the development of those analytical methods as zebrafish is a complex matrix.¹⁶

In this study, we aimed to develop a LC-MS/MS analytical method to quantify the internal concentration of drugs in 5-dpf zebrafish larvae using methamphetamine as a prototypical compound to study whole-body uptake and elimination of methamphetamine as well as uptake and elimination in head and trunk separately. We optimized a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) technique for sample extraction and purification.

Methods

Drugs and chemicals

(+)-Methamphetamine hydrochloride was purchased from Sigma-Aldrich. Acetonitrile (ACN) and formic acid were purchased from Sigma-Aldrich; methanol and ammonium formate from Fisher Chemical; and anhydrous sodium acetate and magnesium sulfate from Molecular Sigma Biology. All solvents used were high-performance liquid chromatography grade.

Zebrafish

Wild-type (AB) adult zebrafish (ZIRC) were housed in the Center for Drug Design and Development (CD3) zebrafish laboratory, University of Toledo. They were housed separately by sex with a density of 10 fish per 7.5L tank containing plants as environmental enrichment and using a water recirculation system (Tecniplast^®; Buguggiate, Italy) to maintain water quality. The room was kept on a 14-h light–10-h dark cycle, and the room and water temperature were maintained at 26°C–28°C. Fish were fed with brine shrimp as the primary food source with flake food (Daniolab, Zebrafish Research Diet, size 4) as a dietary supplement. These adult zebrafish were used for breeding the larval zebrafish needed for experiments. For breeding, adult zebrafish were put into breeding tanks with a ratio of 2 males to 3 females. Embryos were collected the following day and transferred to glass Petri dishes containing egg water (H₂O containing 60 mg/L Instant Ocean^®). Dead embryos and debris were removed, and egg water was changed daily to ensure the optimal conditions for embryo development. The embryos were raised in an incubator with the temperature set at 28.5°C until 5-dpf.

Analytical method development

LC-MS/MS analysis

A Waters system consisting of triple quadrupole combined with a degasser and autosampler using an electrospray ionization source allowed for the analysis. The chromatographic separation of methamphetamine was done on an x-terra C18 column (150 × 2.5 × 5) at a flow rate of 0.3 mL/min. The gradient method was optimized using 5 mM ammonium formate with 0.1% formic acid and methanol with 0.1% formic acid as mobile phases. The injection volume was 10 μL. Optimization of the MS/MS condition, parent and daughter ion identification, and selection of cone voltage and collision energy were performed in positive ion mode. After optimizing cone voltage and collision energy, two m/z transitions were selected for methamphetamine and day-5 methamphetamine to quantify and confirm the analytes.

Sample preparations procedure for zebrafish samples (QuEChERS technique)

This study chose this method based on prior results obtained using the QuEChERS technique to prepare adult zebrafish samples. After the treated zebrafish larvae were euthanized, they were collected in 1.5-mL Eppendorf^® Safe-Lock^® tubes and stored at −4°C. On the day of analysis, the QuEChERS technique was used for the sample preparation before the chromatographic separation. Two micrograms/mL of internal standard solution was prepared in ACN solvent. Then, 250 μL of 2 μg/mL of internal standard solution was used to homogenize 10 euthanized larvae. Homogenization was done manually. Fifty milligrams of anhydrous magnesium sulfate and 12.5 mg of sodium acetate were added to the solution, vortexed for 5 min, followed by centrifugation at 10,000 g for 1 min. Finally, 100 μL of supernatant was diluted with water to make a 50:50 ACN:water solution directly injected into the LC-MS/MS.

To determine recovery, untreated zebrafish larvae were used, with the samples spiked with known concentrations of methamphetamine during the initial step of sample preparation. Concentrations were calculated using the standard calibration curve and compared with the theoretical attention to find the recovery percentage.

Preparation of calibration curve

Samples for each calibration point were prepared by spiking blank zebrafish matrix with methamphetamine hydrochloride at 8 concentrations ranging from 0.06 to 3.9 μg/mL. Seventy-five microliters of 2 μg/mL day-5 methamphetamine (internal standard) was used in all calibration points to make the final volume 250 μL; each injected three times. A matrix-matched calibration curve was prepared, and all the calibration points underwent a similar sample preparation method using the QuEChERS technique.

Limit of quantification

Limit of quantification (LOQ) is the lowest concentration, which was calculated based on the signal-to-noise (S/N) ratio (S/N ≥10).

Specificity and precision

The specificity of the method was evaluated by comparing chromatograms obtained from blank extraction solvent, blank zebrafish samples, and zebrafish samples spiked with methamphetamine.

Interday and Intraday precision were determined. Three different concentrations (LOQ, mid-range, and high concentration) were prepared for three consecutive days, and each was injected three times (n = 3).

Experimental design

Uptake, distribution, and elimination study

Uptake study

For the uptake analysis, 5-dpf larvae were exposed to 100, 150, and 250 μM solution of methamphetamine, in 24-well plate and euthanized at different time points (0.25, 0.5, 1, 2, 3, 6, 12, and 24 h). Drug solutions were prepared in egg water. During the exposure period, the room was kept on a 14-h light–10-h dark cycle, and the room and water temperature were maintained at 26°C–28°C without feeding. Before euthanization, the exposed larvae were washed with egg water to remove the drug adhering to the skin. Washing validation was done before conducting the uptake experiment. At each time point, 10 larvae were pooled, in three replicates in three replicates euthanized by snap freezing using dry ice, and stored at −80°C.

On the analysis day, fish samples were defrosted, and sample preparation was done using the QuEChERS technique. The selection of exposure concentration was based on our preliminary study. Our preliminary study showed that maximal nonlethal concentration (MNLC) at physiologically preferred temperature was 1 mM and the highest concentration with 5-dpf zebrafish performing correctional tendencies to return to normal swimming behavior was 500 μM.¹⁷

Therefore, three different concentrations of methamphetamine were chosen to study concentration-dependent absorption and elimination patterns that were less than the MNLC.

Elimination study

To study elimination, zebrafish larvae were transferred into a drug-free medium after 24-h exposure to 100, 150, and 250 μM of methamphetamine and collected at 1, 2, 3, 6, 8, 12, 24, 30, and 48 h of depuration following the same procedure as uptake.

The 24-h exposure time was selected based on an uptake study where the absorption pattern showed a steady state at this time.

Distribution of methamphetamine in the head and trunk

Based on the data obtained from the absorption and elimination studies, time points were chosen for the distribution study, and internal concentration was determined separately in the head and trunk. Pools of 10 5-dpf zebrafish larvae were exposed to drug solutions as described for the absorption and elimination studies. For the absorption study, at 0.25, 3, 6, 12, and 24 h of exposure, they were euthanized, and the head and trunk were separated and for the elimination study, after exposure for 24 h, they were euthanized at 0.25, 1, 2, 8, and 12 h after being transferred into drug-free media. The head and trunk were separated and stored in Eppendorf tubes at −80°C. Samples were prepared as described above using the QuEChERS method.

Nonlinear mixed-effects modeling

Nonlinear mixed-effects modeling was performed on the data using the Phoenix™ Pharmacokinetic and Pharmacodynamic (PK/PD) Platform Version 8.3.

Data analyses

All data analyses were performed using Prism GraphPad 5.0 (San Diego, CA, USA). The comparison between the concentration of methamphetamine in the head and trunk was done using a t-test. The significance level for all analyses was set at p < 0.05.

Results and Discussion

LC-MS/MS analysis

The separation method for methamphetamine was obtained by the gradient method, using 100% 5 mM ammonium formate for the first 1 min, followed by 60% methanol with 0.1% formic acid for 1.5 min, which increased to 95% for the next 2.5 min equilibrated by 100% 5 mM ammonium formate with 0.1% formic acid. So, the total run time was 10 min at a flow rate of 0.3 mL/min with a 10 μL injection volume. The chromatogram obtained under this condition is shown in Supplementary Figure S1. The cone voltage and collision energy were set at 30 V and 20 KV, respectively (Table 1). Multiple reaction monitoring transitions were acquired for methamphetamine (150.14 > 91.01, 150.14 > 119.12) and day-5 methamphetamine (155.1 > 121.25, 155.1 > 92.09).

Table 1.

Analyte Transition Ions With a Suggested Indication of Their Formation, Associated Collision Energy, Cone Voltage, and Ionization Mode

Compound	Transition (m/z)	Collision energy (Kv)	Cone voltage (V)	Ionization mode
		20	30	+
		20	30	+

Data acquisition and peak processing were performed using Masslynx software version 4.1 (Waters). The quantification of methamphetamine was done by correcting the peak area response of methamphetamine with the peak area response of the day-5 methamphetamine (internal standard) (Table 1). All calculations concerning the evaluation of recorded data were made in excel and GraphPad prism.

Matrix-matched calibration curve

The matrix-matched standard calibration curve was prepared, which showed good linearity over a concentration range of 0.06–3.8 μg/mL (r² = 0.9991; Supplementary Fig. S2). This calibration curve was used for further uptake and elimination analysis calculation.

Limit of quantification

The lower LOQ was 60 ng/mL, which is higher than the values reported in the literature, for example, 5 ng/mL¹⁸ and 28 ng/mL.¹⁹

Specificity, precision, and accuracy

No interfering peaks are present at the retention time of methamphetamine, demonstrating the method's specificity (Supplementary Fig. S3). The obtained relative standard deviation (RSD) values were 7.9%, 9.6%, and 14.3% for a lower, middle, and higher LOQ, respectively, for interday precision. The obtained RSD values were 19.47%, 4.9%, and 5.6%, respectively, for intraday precision. These results comply with the regulatory guidelines on bioanalytical method validation.²⁰

Validation of sample preparation procedure

The QuEChERS technique is used for the extraction and purification of samples. The recovery percentage found was 92% to 120%. Recoveries ranging between 80% and 120% are generally acceptable for sample preparation procedures.²⁰

Uptake and elimination analysis

An uptake study was done to understand how methamphetamine is taken up by zebrafish larvae from the exposure medium (Fig. 1). Our study showed that methamphetamine was quantifiable in whole-body homogenates of 5-dpf zebrafish larvae as early as 15 min after exposure for the two higher exposure concentrations. The absorption pattern was quite complex for highest exposure concentration (250 μM). The internal concentration continued to increase up to 5 h, followed by an initial plateau, with a second plateau occurring after 8 h. Thus, absorption seems biphasic with a half-life of 8.27 h. It looks like the combination of two zero-order absorption processes, one with fast absorption but short duration and the other with slow absorption that lasts longer. Nevertheless, concentration absorption gradually increases with time overall for the lower two exposure concentrations (100 and 150 μM). The internal concentration of methamphetamine continuously increased for several hours with continuous drug medium exposure, that is, waterborne route. The internal concentration of methamphetamine was higher for higher exposure concentrations.

FIG. 1.

Uptake and elimination study of methamphetamine in 5-dpf zebrafish larvae. Larvae were exposed to 250, 150, and 100 μM concentrations of methamphetamine. For the elimination study, larvae were exposed at 5-dpf for 24 h and then transferred to drug-free egg water for up to 48 h. Methamphetamine was quantified in whole-body homogenates by liquid chromatography–tandem mass spectrometry. 5-dpf, 5-day postfertilization.

The elimination study showed that upon transfer to a drug-free medium, zebrafish larvae showed a biphasic elimination pattern with an overall elimination rate of 0.084 h⁻¹ and clearance with 3.13 μmol/h × μg/mL. Zebrafish larvae eliminated about 50% of the absorbed methamphetamine within 6 h upon transfer to a drug-free medium when exposed to 250 and 150 μM methamphetamine. These results are similar to an elimination study done on cocaine in zebrafish larvae.¹²

After 24 h of transfer into drug-free media, the internal concentration was not able to be quantified by this method. However, when exposed to 100 μM methamphetamine, only 36% were eliminated within 12 h, indicating slow clearance. This may suggest that a gradient-dependent diffusion might influence the elimination process in larvae. It is speculated that the biphasic elimination pattern is a partial elimination due to a particular spatial distribution and passive diffusion.²¹ Methamphetamine accumulates more in the head than in the trunk. However, it was found that methamphetamine accumulates first in the trunk. When quantified, the internal concentration of methamphetamine was found to be greater in the trunk after 15 min of exposure.

Most zebrafish experiments rely on water exposure and drug absorption primarily by skin or gill diffusion.²² When methamphetamine enters the body, it enters the systemic circulation, which is why the concentration in the trunk is greater initially. However, with the increase in exposure time, methamphetamine accumulates more in the head region. The higher concentration of methamphetamine in the head region over time may indicate that the functional blood–brain barrier is lacking at this stage. This is consistent with previous research that reported limited blood–brain barrier development in zebrafish larvae up to 9- to 10-dpf.^12,23

In addition, Fleming et al. examined drug concentration in the head and trunk tissues of 5-dpf zebrafish larvae for a panel of 5 medications known to cross or not cross the blood–brain barrier in mammals. All drugs were equally distributed, indicating the limited development of the blood–brain barrier at this age.²⁴ After exposure to 250 μM methamphetamine, the internal concentration of methamphetamine in the trunk was significantly higher than in the head after 15 min of exposure. With increased exposure time, methamphetamine gradually accumulated in the head region and was higher after 3 h of exposure.

A significantly higher concentration was seen after the 6 h of the exposure period (Fig. 2). This trend was similar when zebrafish larvae were exposed to a 150 μM concentration of methamphetamine (Fig. 2). But unlike when exposed to 250 μM concentration of methamphetamine, the internal concentration was not quantifiable after 15 min of exposure. However, when exposed to a lower concentration (100 μM), the internal concentration of methamphetamine could not be quantified in either the head or trunk, likely because the methamphetamine level was below the limit of detection of our LC-MS/MS method.

FIG. 2.

Uptake and elimination study in the head and trunk of 5-dpf zebrafish. (A) Bar graph showing a comparison of uptake by head and trunk separately when exposed to 150 μM of methamphetamine. (B) Bar graph showing comparison of uptake by head and trunk separately when exposed to 250 μM of methamphetamine. *Significant level p < 0.05. **Significant level p < 0.01. ***Significant level p < 0.001. (C) Uptake and elimination study in the head and trunk of 5-dpf zebrafish larvae when exposed to 150 μM. (D) Uptake and elimination study in the head and trunk of 5-dpf zebrafish larvae when exposed to 250 μM methamphetamine.

Although the initial elimination percentage is similar in the head and trunk, that is, 33.7% and 36.4% elimination in the trunk region and head region within 3 h of depuration, respectively, the elimination study showed that methamphetamine eliminated from the trunk faster than the head. Thus, methamphetamine was retained more in the head than in the trunk. This result was consistent with findings from earlier studies finding that basic drug (cocaine and meta-chlorophenylpiperazine) binds to melanin present in the eye of zebrafish larvae, which is highly pigmented, which is the primary reason for higher accumulation and retention of drug in the head.^12,13 The internal concentration upon transfer to a drug-free medium was not able to quantify levels of methamphetamine in the trunk after 3 h when exposed to 250 μM methamphetamine or after 15 min when exposed to 150 μM methamphetamine. However, the internal concentration in the head was quantifiable at 6 and 3 h when exposed to 250 and 150 μM solutions, respectively (Fig. 2).

Conclusion

An effective method for quantifying methamphetamine in zebrafish larvae was developed using LC-MS/MS to determine pharmacokinetic and toxicokinetic properties. The QuEChERS technique was an effective sample preparation and purification method for zebrafish larvae and provided high recovery percentages. This approach can therefore be used to relate tissue levels to pharmacological and toxicological outcomes, including behavioral studies. The accumulation of methamphetamine was higher in the head region than the trunk with increased exposure time, which may be an indication of a lack of functional development of the blood–brain barrier in 5-dpf zebrafish larvae, an indication of active brain transport, or accumulation in the brain by binding to melanin. This latter explanation is consistent with the slow elimination of methamphetamine from the head, suggests increased retention in the head, which might be related to methamphetamine binding to melanin in the eye.

Ethics Statement

This study was approved by the Institutional Animal Care and Use Committee (IACUC). The protocol for zebrafish breeding, embryo production, and zebrafish husbandry were approved under IACUC protocol number 400099-UT titled “UT-CD3: Zebrafish Breeding for Population Maintenance and Embryo Production.” Similarly, the protocol for methamphetamine exposure was approved under IACUC protocol number: 105414-UT titled “Chemical Exposure and Development in the zebrafish, Danio rerio.”

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by a grant from the National Institute on Drug Abuse (U01DA054330).

Supplementary Material

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Supplementary Material

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