научная статья по теме SEPARATION OF SARCOSINE AND L-ALANINE ISOMERS USING CORONA DISCHARGE ION MOBILITY SPECTROMETRY Химия

Текст научной статьи на тему «SEPARATION OF SARCOSINE AND L-ALANINE ISOMERS USING CORONA DISCHARGE ION MOBILITY SPECTROMETRY»

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SEPARATION OF SARCOSINE AND L-ALANINE ISOMERS USING CORONA DISCHARGE ION MOBILITY SPECTROMETRY © 2014 Shiva Mirmahdieh, Taghi Khayamian

Department of chemistry, Isfahan university of technology Isfahan, 84156-8311, Iran Received 15.02.2012; in final form 15.11.2012

The capability of corona discharge ion mobility spectrometry (CD-IMS) for separation and quantification of sarcosine and L-alanine isomers has been evaluated for the first time. Although these two compounds have the same mass and m/z values in mass spectrometer, ion mobility spectrometry was able to separate and determine them. Variables including carrier gas flow rate, injection and cell temperatures were optimized. The reduced mobilities (K0) of sarcosine and L-alanine were 1.96 and 1.83, respectively, based on the reduced mobility of nicotinamide. At the optimized conditions the detection limit of sarcosine and L-alanine were 0.7 and 0.9 ^g/mL, respectively. The relative standard deviation (RSD) was found to be 6%. Furthermore, a sample injection port of a gas chromatograph was also modified to introduce solvent-free samples into the IMS.

Keywords: ion mobility spectrometry, sarcosine, L-alanine.

DOI: 10.7868/S0044450214060103

Sarcosine, an N-methyl derivative of the amino acid glycine, is an intermediate and byproduct in glycine synthesis and degradation. Sarcosine is identified as a key metabolite, whose concentration increases greatly during prostate cancer progression to metastasis and could be detected in urine [1]. Concentration ratio of sarcosine to L-alanine was found to be significantly higher in urine derived from biopsy-positive prostate cancer patients compared with biopsy-negative controls [2]. These two isomers have the same fragment at m/z 44 in mass spectrometer and even MS/MS does not discriminate them in a mixture, and a separation technique is required [2, 3]. The ion at this m/z corresponds to the immonium ion derived by loss of water and carbon monoxide. Therefore, determination of these two isomers requires a separation technique. These compounds have been determined by GC—MS [1], HPLC/ESI—MS/MS [4] and also LC-MS/MS [5]. In addition, differential mobility analysis-mass spectrometry (DMA—MS) with peak deconvolution has also been reported for the determination of these compounds [2].

Ion mobility spectrometry (IMS) is an analytical technique that separates gas-phase ions at atmospheric pressure. IMS is a fast, low-cost, and sensitive method for the detection of trace quantities of volatile organic and inorganic compounds [6]. Moreover, IMS is not only a simple detector, it can also be used to separate ionic species based on their mobilities [7]. This ion mobility depends on mass, charge state, and shape of the ion. The ion mobility coefficient can be ob-

tained using the drift velocity of ions in an electrical field [7, 8]. The expression for the drift velocity vd is given by the following equation:

v d = KE,

where K is the ion mobility coefficient and E — the electric field.

The ion mobility depends on temperature and pressure, and the temperature and pressure effects are normalized to standard conditions to compare values of K in different laboratories through the use of the reduced mobility constant (K0, cm2/V s):

K0 = K (27^ T)( P/760),

where T is temperature in Kelvin and P is pressure in torr of the gas atmosphere through which the ions move. When all the instrumental parameters are controlled, the mobility coefficient depends on the size-to-charge ratio and the reduced mass of the ion in the supporting atmosphere. However, ions of the same mass but different functional groups, or ions of the same functional group but different geometrical arrangements (isomers), often exhibit different K0 values, reflecting the dependence of mobility on the shape and size. The expression for the K is given by the following equation:

K _ 3e (2n)1/2 (1 + a) _ 16N(kTf ) Qd (Tef )

IMS operation parameters

Operating parameter Setting

Corona voltage 10.5 kV

Counter electrode voltage 7.5 kV

Drift flow gas (N2) 600 mL/min

Carrier flow gas (N2) 500 mL/min

Cell temperature 170°C

Injection port temperature 180°C

Shutter grid pulse 100

Drift field 500 V/cm

Drift tube length 11 cm

where e — is the charge of electron; N — is the number density of drift gas at the pressure of measurement; a — is the correction factor; | — is the reduced mass of ion and gas of the supporting atmosphere; Tef — is the effective temperature of the ion determined by thermal energy and the energy acquired in the electric field; — is the effective collision cross section of the ion in the supporting atmosphere and k — is Boltz-mann's constant. The mass of an ion strongly affects the K0, also mobility depends on the ion structure and the collision cross section. Ion mobility is able to distinguish between isomeric compounds with identical mass-to-charge ratios (m/z), which cannot be separated from each other using mass spectrometry [9].

The objective of this study is to investigate the ability of the CD—IMS to separate sarcosine and L-ala-nine isomers. Furthermore, sample introduction system [10] was also modified to introduced solvent-free samples into the CD—IMS. Finally, at the optimized conditions analytical parameters of the compounds were obtained.

EXPERIMENTAL

Chemicals and reagents. Sarcosine and L-alanine were purchased from Merck (Darmstadt, Germany). Other chemicals were of analytical reagent grade and obtained from Merck. Deionized water was prepared by OES (Overseas Equipment & Services) water purification system (OK, USA). Stock standard solutions of each analyte were prepared separately in metha-nol—formic acid 98 : 2 (v/v) at 1000 mg/L and stored at 4°C.

Instrumentation. The IMS apparatus with CD as the ionization source in the positive mode has been described previously. Liquid samples (in methanol solvent) were injected into the IMS [10]. In this work, since ion peak of sarcosine overlaps with methanol peak, a solvent-free sample introduction system was de-

veloped. A Teflon screw cap (outside diameter: 28 mm, inner diameter: 9.3 mm and height: 17.6 mm) holding a fine stainless steel filament was used to introduce samples. A sample (10 ^L) was placed on the filament, and after evaporating the solvent the cap was inserted into the injection port. The injection port consists of a cubic shape of 60 mm x 50 mm x 25 mm dimensions. A cartridge heater, 250 W, was used for heating the port. The inlet and outlet of the carrier gases were positioned on opposite sides at top and bottom of the sampling chamber, respectively. The sample gas was introduced into the cell via a brass tube (i.d. 3 mm and o.d. 6 mm), positioned orthogonal to the corona needle. This tube was also used as the counter electrode. The brass tube and an aluminum ring around the IMS cell were connected to ensure identical potentials. In the reaction region of the IMS cell the analyte molecules were ionized in ion-molecular exchange reactions with reactant ions. These ions were injected electronically from the ion source region into the drift region by means of an ion gate. In the drift region ions were separated based on their mobilities. Nitrogen was used as the drift and carrier gas. The schematic diagram of the IMS cell is shown in Fig. 1. All IMS spectra were obtained by data acquisition software and each IMS spectrum was the average of 50 individual spectra.

RESULTS AND DISCUSSION

Ion mobility spectra. The individual ion mobility spectra of sarcosine and L-alanine with the binary mixture of these compounds are shown in Fig. 2. These spectra were obtained under the optimized IMS conditions given in Table. In this work nicotinamide with the reduced mobility (K0) value of1.85 cm2/V s was used as an external standard for the instrument [11], and, therefore, the K0 values of 1.96 and 1.83 were calculated for sarcosine and L-alanine main peaks, respectively. The K0 of1.98 cm2/V s for sarcosine and 1.82 cm2/V s for P-alanine in N2 were obtained by Johnson and coworkers using ESI—IMS [12]. Also, measured mobilities for sarcosine and L-alanine in air by Martínez—Lozano and coworkers using DMA—MS were 1.94 and 1.89, respectively, and the method was unable to separate these two isomers peaks and the deconvolution technique was required for their separation [2]. The ion-ization sources for the mentioned techniques were electrospray. This is the first work using corona discharge as an ionization source for these compounds.

Effect of injection port temperature and carrier gas flow rate. The influence of injection port temperature on spectra of sarcosine and L-alanine was evaluated. Experimental results are presented in Fig. 3. According to these results, by increasing injection port temperature from 160 to 190°C the analyte responses increased, however at higher temperature thermal breakdown of compounds could occur, yielding a lower intensity of ion mobility peaks. Therefore, the injection

SEPARATION OF SARCOSINE AND L-ALANINE ISOMERS USING CORONA

575

(a) Г

+ Power Supply Power Supply -

(A) (B)

Exit

Shutter Grid

L

Drift Gas L

. J

Counter Electrode

Conductor Rings

_Q

Signal

Aperture Grid

t= -«—Carrier Gas

(b)

28 mm

Injection port

25 mm

50 mm

Fig. 1. Schematic diagram of the CD—IMS with the sample introduction system used in this work: (a) CD—IMS, (b) teflon screw cap holding a fine stainless steel filament for introducing the sample.

port temperature of 180°C was selected for the experiments.

To determine the influence of the carrier gas flow rate on the intensity of ion mobility peaks, the flow rate was varied from 150 to 550 mL/min. According to the obtained results (Fig. 4), the flow rate of500 mL/min was selected and used for further studies.

Effect of drift tube temperature on resolution. The

resolution of IMS is measured in terms of "peak-to-peak resolution" (Rpp) and defined on the basis of separation of pa

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