Optimized Isomer Separation of Synthetic Cathinones by LC-MS/MS

• Ensure crucial isomer separations of synthetic cathinones that are not easily separated with conventional C18 columns.
• Overcomes the challenge of not being able to fully separate and quantify isobaric isomers by MS/MS.
• Unique stationary phase provides good retention of aromatic compounds, resulting in sharper peak shapes.

In the last decade, synthetic cathinones have become widespread in recreational drug use, prompting a need for forensic toxicology laboratories to develop reliable workflows for their identification and quantification. Synthetic cathinones are derivatives of the naturally occurring compound cathinone and are a unique class of psycho-stimulants. They have an aromatic structure and contain structurally similar isomers that are challenging to separate using conventional C18 columns. A reliable analysis that achieves isomer separation is crucial because the pharmacological and toxicological properties of the various isomers differ significantly. Since the respective ortho, meta, and para isomers of methylmethcathinone (MMC) and methylethcathinone (MEC) are isobaric, they are not easily separated and quantified by MS/MS.

Here, we show the analysis of synthetic cathinone in serum samples on the Raptor Biphenyl core-shell column by LC-MS/MS analysis. The phase of this Raptor column exhibits strong pi-interactions, making it especially selective to the separation of the differently substituted aromatic rings that make up these isomers. A deuterated internal standard (butylone-d3) was chosen for use due to its availability and cost savings. Using the method conditions specified below, accurate quantitative results and reliable separations were achieved for the synthetic cathinone isomers of MMC and MEC, in serum samples. Figure 1 shows a section of the chromatogram from a spiked serum sample at 100 ng/mL to illustrate the isomer separations. Figure 2 shows a spiked serum sample at the limit of quantification, 5 ng/mL.

Figure 1: A spiked serum sample at 100 ng/mL shows excellent isomer separation.

LC_CF0790

Peaks

	Peaks	tR (min)	Parent Ion	Product Ion 1	Product Ion 2
1.	2-MMC	5.75	178.1	145.1	160.0
2.	3-MMC	6.15	178.1	145.1	160.0
3.	4-MMC	6.70	178.1	145.1	160.0
4.	Butylone-D3	7.25	225.1	176.9
5.	2-MEC	7.77	192.1	174.0	144.0
6.	3-MEC	8.30	192.1	174.0	144.0
7.	4-MEC	8.71	192.1	174.0	144.0

Conditions

Column	Raptor Biphenyl (cat.# 9309A12)
Dimensions:	100 mm x 2.1 mm ID
Particle Size:	2.7 µm
Pore Size:	90 Å
Temp.:	50 °C
Standard/Sample
Diluent:	Water
Conc.:	100 ng/mL
Inj. Vol.:	10 µL
Mobile Phase
A:	Water:methanol 95:5, 0.1% formic acid
B:	Methanol, 0.1% formic acid
	Time (min) Flow (mL/min) %A %B 0.00 0.5 95 5 1.00 0.5 95 5 12.00 0.5 83 17 12.01 0.5 2 98 20.00 0.5 2 98 20.01 0.5 95 5 23.00 0.5 95 5

Detector	SCIEX API 4000 QTRAP MS
Ion Mode:	ESI+
Instrument	Shimazdu LC 20
Sample Preparation	200 µL of serum was spiked with 10 µL internal standard (butylone-D3, 1 µg/mL) and prepared to a concentration of 100 ng/mL with a stock containing the 6 synthetic cathinones. A protein precipitation was performed with 200 µL methanol. Samples were vortexed and centrifuged for 8 minutes at 1625 rpm. 50 µL of the supernatant was diluted with 150 µL water and transferred to a vial for analysis.
Notes	An additional equilibration time of 5 minutes was added before each injection to ensure stable retention times.

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Figure 2: Spike serum sample at the LOQ, 5 ng/mL.

LC_CF0789

Peaks

	Peaks	tR (min)	Parent Ion	Product Ion 1	Product Ion 2
1.	2-MMC	5.95	178.1	145.1	160.0
2.	3-MMC	6.25	178.1	145.1	160.0
3.	4-MMC	6.76	178.1	145.1	160.0
4.	Butylone-D3	7.45	225.1	176.9	–
5.	2-MEC	7.99	192.1	174.0	144.0
6.	3-MEC	8.38	192.1	174.0	144.0
7.	4-MEC	8.90	192.1	174.0	144.0

Conditions

Column	Raptor Biphenyl (cat.# 9309A12)
Dimensions:	100 mm x 2.1 mm ID
Particle Size:	2.7 µm
Pore Size:	90 Å
Temp.:	50 °C
Standard/Sample
Diluent:	Water
Conc.:	5 ng/mL
Inj. Vol.:	10 µL
Mobile Phase
A:	Water:methanol 95:5, 0.1% formic acid
B:	Methanol, 0.1% formic acid
	Time (min) Flow (mL/min) %A %B 0.00 0.5 95 5 1.00 0.5 95 5 12.00 0.5 83 17 12.01 0.5 2 98 20.00 0.5 2 98 20.01 0.5 95 5 23.00 0.5 95 5

Detector	SCIEX API 4000 QTRAP MS
Ion Mode:	ESI+
Instrument	Shimazdu LC 20
Sample Preparation	200 µL of serum was spiked with 10 µL internal standard (butylone-D3, 1 µg/mL) and prepared to a concentration of 5 ng/mL with a stock containing the 6 synthetic cathinones. A protein precipitation was performed with 200 µL methanol. Samples were vortexed and centrifuged for 8 minutes at 1625 rpm. 50 µL of the supernatant was diluted with 150 µL water and transferred to a vial for analysis.
Notes	The higher concentration internal standard peak is not shown in order to focus on the lower concentration target analytes. An additional equilibration time of 5 minutes was added before each injection to ensure stable retention times.

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Acknowledgements:

Data courtesy of Alexandra Maas, Ph.D., University of Bonn. Thank you to Mrs. Maas for allowing us to use this data.

References:

A. Maas, et al., Separation of ortho, meta and para isomers of methylmethcathinone (MMC) and methylethcathinone (MEC) using LC-ESI-MS/MS: Application to forensic serum samples, J. Chromatogr. B (2017), http://dx.doi.org/10.1016/j.jchromb.2017.01.046

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