Quantitative Mass Spectrometric Determination of Methylphenidate Concentration in Urine using an Electrospray Ionization Source Integrated with a Polymer Microchip

Yanou Yang, Jun Kameoka, Timothy Wachs, Jack D. Henion, H. G. Craighead

We have demonstrated the use of a simple microfabricated electrospray ionization source for coupling microfluidic chips to mass spectrometry (MS). A polymer-based microchip, coupled to a triple quadrupole mass spectrometer, has been employed for direct infusion quantitative bioanalysis of methylphenidate (Ritalin) extracted from human urine samples. The approach used a microfabricated polymer electrospray emitter to couple a microfluidic channel to a stable electrospray ionization source. The microchip was fabricated from cyclo-olefin plastic plate by hot embossing and thermal bonding. This microfluidic chip contained two independent microfluidic channels, integrated with two corresponding electrospray emitters and an internal gold electrode. Liquid-liquid extraction was used to prepare urine samples, spiked with methylphenidate. A trideuterated analogue of methylphenidate (methylphenidate-d₃) was used as the internal standard for the analysis. The system showed good electrospray stability, and reproducibility with different spray tips. Four different electrospray tips were used to analyze the same sample, and the results showed very small variation with a RSD of 1.4%. A standard curve prepared for methylphenidate in urine (R²=0.99) was linear over the range of 0.4-800ng/ml. The precision of the quality control (QC) samples for three different concentrations ranged from 19.1% at 20ng/ml, 3.17% at 200ng/ml to 3.45% at 667ng/ml while the accuracy was 96.3% at 20ng/ml, 101.2% at 200ng/ml and 101.6% at 667ng/ml. No system carryover was detected even when the same device was used for sequential analysis. These results suggest the potential of this microdevice for MS-based quantitative analysis in drug discovery and development.

Figure 1: Device fabrication. A: Gold electrode fabrication. (1) photoresist spinning; (2) photolithography; (3) gold evaporation; (4) lift-off. a: photoresist; b: cyclo-olefin polymer plate; c: gold film. B: fabrication process of the device with two electrospray tips by thermal bonding. a: top plastic chip with embossed fluidic channel; b: reservoirs; c: microfluidic channel; d: parylene film with two electrospray tips; e: bottom plastic chip with fabricated electrode; f: gold electrode; g: hole to fit a metal screw.

Figure 2: Configuration of microchip interfacing to API-III plus triple quadrupole mass spectrometer (not drawn to scale). a: microchip; b: syringe pump; c: power supply; d: x, y, z stage.

Figure 3: Fabricated devices: A: Array of two parylene electrospray tips with a distance of 1mm. B: Picture of the microchip for interfacing to mass spectrometer (3cmx2cm, 4 mm thick).

m/z 237.2 > m/z 84.2

RSD=3.05%

100%=7.79 X 10⁴ cps

Figure 4: Selected reaction monitoring (m/z 237.2> m/z 84.1) ion current for extracted urine sample containing 150ng/ml methylphenidate-d₃.

B

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Figure 5: SRM ion current of the same extracted urine sample containing methylphenidate and methylphenidate-d₃ using four different electrospray tips. A: tip 1 from chip 1, I/I₀=0.470, acquisition time 30s; B: tip 1 from chip 2, I/I₀=0.463, acquisition time 30s; C tip 2 from chip 2, I/I₀=0.460, acquisition time of 25s; D: tip 1 from chip 3, I/I₀=0.457, acquisition time =20s.

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Figure 6: SRM ion current for calibration standards and QC sample in urine. A: 1.6ng/ml methylphenidate, I/I₀=0.0447, acquisition time 30s B: 10ng/ml methylphenidate, I/I₀=0.11, acquisition time 30s; C: 200ng/ml methylphenidate, I/I₀=1.51, acquisition time 34s; D: 800ng/ml methylphenidate, I/I₀=5.50, acquisition time 18s.

B

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Figure 7: SRM ion current of the blank sample for methylphenidate analyzed with one electrospray tip indicating the signal obtained before and after running 800ng/ml calibration standards. A: Before running the highest level of calibration standard, I/I₀=0.0312, acquisition time 30s B: After running the highest level of calibration standard. I/I₀=0.0314, acquisition time 30s.

Publication Reference:

"Quantitative Mass Spectrometric Determination of Methylphenidate Concentration in Urine Using an Electrospray Ionization Source Integrated with a Polymer Microchip", Yanou Yang, Jun Kameoka, Timothy Wachs, Jack D. Henion, and H. G. Craighead, Analytical Chemistry, Web Release April 3, 2004.