Simultaneous Determination of Mephedrone, Methylone, MDPV, and

Amphetamines in Urine by LC/MS/MS Amanda Rigdon1*, Mike Coyer2, Jack Cochran1, Ty Kahler1, and Paul Kennedy3.

(1)Restek. Bellefonte, PA, USA. (2) Northern Tier Research, Mayfield, PA, USA. (3)Cayman Chemical, Ann Arbor, MI, USA.

Introduction

Abuse of substances marketed as ‘research chemicals’ often sold

for research purposes only or added to consumer products labeled

‘not for human consumption’ has become increasingly popular.

Cathinones, including mephedrone, methylone, and MDPV, are one

class of compounds that have appeared on the market as part of the

‘research chemical’ movement. These compounds are commonly

sold as bath salts, however drug users often snort or ingest these

compounds to induce an amphetamine-like high. (Figure 1). In

September 2011, mephedrone, methylone, and MDPV were placed

on the DEA Schedule I list on an emergency basis. Because of this

scheduling, demand for testing will increase.

Figure 1: Photos of Bath Salts (left) and ‘Research Chemicals’ (right)

Chromatographic Method Development and Validation:

Because these compounds are very similar to amphetamines, and

are used either as a substitute for or in conjunction with

amphetamines [1], a chromatographic method was developed to

detect both amphetamines and cathinones in a single run. In

addition to the compounds mentioned in the title of this poster,

several additional ‘research chemicals’ marketed to be similar to

MDPV were included in the method. The final chromatographic

method and results are detailed in Figure 2.

Figure 2: 100 ng/mL Spiked Urine Sample

Table 1: Peak List and Transitions Used

Method Performance:

A partial validation was performed on the three cathinones and

amphetamines listed in the title of this work. Preliminary data for the

remaining compounds was also collected, and is available upon

request. LOD/LOQ, linearity, precision, and accuracy data were

collected and are shown in Table 2. Note that at the time this work

was performed, only mephedrone and its deuterated analog were

available as certified reference standards. In order to evaluate the

method for the remainder of the compound list, the ‘research

chemicals’ shown in Figure 1 were used as mock reference standards

for this work. Although a thorough characterization was not performed,

all substances were analyzed for purity using LC-MS, and no

additional compounds from 100 – 1000m/z were detected in the

products. All samples were analyzed on a Shimadzu UFLCXR equipped with

an AB SCIEX API 4000 MS/MS. Rather than being extracted, samples were

prepared using a dilute-and-shoot methodology. All samples were diluted 10x

in starting mobile phase containing 30 ng/mL internal standard.

Table 2: Method Performance Summary

Discussion:

All values in the table above reflect results from the quantifier ion.

MDA, MDMA, and MDEA were not evaluated using external control

samples due to an error in writing the MS/MS method. These three

compounds were evaluated for the remaining parameters at a later

date. The calibration curve for this project was prepared from 1

ng/mL to 500 ng/mL, however, based on recent findings, urine

concentrations of MDPV can range from < 10 ng/mL to > 8000

ng/mL. When samples > 1000 ng/mL were analyzed according to

the method detailed above, both detector and column overloading

were observed. This can be remedied by using either a higher

sample dilution factor and/or a smaller injection volume, however a

range such as this may exceed the linear dynamic range of the

MS/MS detector.

Authentic Sample Analysis:

One authentic sample was

obtained for this study. This

sample was diluted as

described above and analyzed

along with a calibration curve.

MDPV was found at a level of

116 ng/mL in the sample

(Figure 3).

Figure 3: Authentic Sample

Positive for MDPV

Conclusion:

The method developed here is

suitable for the quantitative

analysis of cathinones and

amphetamines in urine at low

levels. Further work needs to be performed to determine

performance with high-level samples. Preliminary data indicates

that this method is also suitable for several of the newer designer

drugs that may become used more widely in the near future due

to the DEA scheduling of several cathinones.

References:

[1] P. Kriikku, L. Wilhelm, O. Schwarz, J. Rintatalo. Forensic Science International. 2011, 210, 1-3; 195 - 200