Research Article Received: 29 July 2010
Revised: 28 October 2010
Accepted: 29 October 2010
Published online in Wiley Online Library: 00 Month 2011
Rapid Commun. Mass Spectrom. 2011, 25, 429–435 (wileyonlinelibrary.com) DOI: 10.1002/rcm.4844
Strong anion exchange liquid chromatographic separation of protein amino acids for natural 13C-abundance determination by isotope ratio mass spectrometry Daniel A. Abaye, Douglas J. Morrison and Tom Preston* Stable Isotope Biochemistry Laboratory, Scottish Universities Environmental Research Centre, Rankine Avenue, Scottish Enterprise Technology Park, East Kilbride, Glasgow G75 0QF, UK
Amino acids are the building blocks of proteins and the analysis of their 13C abundances is greatly simplified by the use of liquid chromatography (LC) systems coupled with isotope ratio mass spectrometry (IRMS) compared with gas chromatography (GC)-based methods. To date, various cation exchange chromatography columns have been employed for amino acid separation. Here, we report strong anion exchange chromatography (SAX) coupled to IRMS with a Liquiface interface for amino acid d13C determination. Mixtures of underivatised amino acids (0.1–0.5 mM) and hydrolysates of representative proteins (prawns and bovine serum albumin) were resolved by LC/IRMS using a SAX column and inorganic eluents. Background inorganic carbon content was minimised through careful preparation of alkaline reagents and use of a pre-injector on-line carbonate removal device. SAX chromatography completely resolved 11 of the 16 expected protein amino acids following acid hydrolysis in underivatised form. Basic and neutral amino acids were resolved with 35 mM NaOH in isocratic mode. Elution of the aromatic and acidic amino acids required a higher hydroxide concentration (180 mM) and a counterion (NOS 3 , 5–25 mM). The total run time was 70 min. The average d13C precision of baseline-resolved peaks was 0.75% (range 0.04 to 1.06%). SAX is a viable alternative to cation chromatography, especially where analysis of basic amino acids is important. The technology shows promise for 13C amino acid analysis in ecology, archaeology, forensic science, nutrition and protein metabolism. Copyright ß 2011 John Wiley & Sons, Ltd.
Recent developments in liquid chromatography/isotope ratio mass spectrometry (LC/IRMS) interface design[1,2] have heralded the precise determination of the d13C values of amino acids at natural abundance without the need for prior derivatisation.[3–6] The principal disadvantage of derivatisation is that additional carbon atoms are added which attenuate the 13C signal and may introduce isotopic fractionation, resulting in error propagation in determining 13 C abundance by gas chromatography/combustion/IRMS (GC/C/IRMS), where derivatisation of amino acids is mandatory.[7] Complete chromatographic resolution of analytes prior to non-fractionating C oxidation to CO2, and subsequent introduction into an IRMS instrument is key to achieving isotope ratio measurement precision and accuracy. To date, various forms of cation exchange chromatography (two-dimensional (2D),[3,8] strong cation exchange[4] and mixed-mode[5,6]) have been reported for 13C amino acid determination by LC/IRMS. The utility of SAX chromatography coupled with IRMS has been reported for the analysis of sugars;[2,9] however, it has not been explored for amino acid analysis, although robust protocols have been developed
* Correspondence to: T. Preston, Stable Isotope Biochemistry Laboratory, Scottish Universities Environmental Research Centre, Rankine Avenue, Scottish Enterprise Technology Park, East Kilbride, Glasgow G75 0QF, UK. E-mail:
[email protected]
for amino acid separation by strong anion chromatography.[10–12] A brief survey of amino acid separations in the liquid phase shows that 2D chromatography, where a strong cation exchange column was placed ahead of a reversed-phase C30 column, separated 11 of the 15 amino acids analysed following acid hydrolysis and the d13C precision of several amino acids was about 0.3%.[3] Similarly, use of a reversed-phase column with ion-pairing reagents separated all 15 amino acids hydrolysed from bone collagen.[8] However, significant isotope separation on the column was reported although the accuracy was within 2% of the expected values. A strong sodium cation exchange column has also been used in LC/IRMS, similar to classical amino acid analysis, and the system performance compared with that of GC/C/IRMS.[4] The d13C precision was similar for both techniques at 10) and are ionisable at high pH. They are, therefore, amenable to separation by anion exchange chromatography. Strong anion columns are commercially available and capable of resolving nearly all protein amino acids. Furthermore, there are situations where strong anion chromatography may confer a distinct advantage, for instance where basic and other clearly resolved amino acids are the target of investigation. Strong anion exchange chromatography coupled with IRMS has been employed to measure13C in neutral[2] and acidic sugars from environmental sources[9] and 13C-ethanol from wine.[13] The isotope ratio precision was reportedly 99.0%), amino acids (98-99% purity), bovine serum albumin (BSA; 98%), and glucose (98%) were purchased from Sigma-Aldrich (Poole, UK). Sodium hydroxide (high purity, 46/48% solution) was purchased from Fisher Scientific (Loughborough, UK). Prawns (exoskeleton removed) were obtained from a local supermarket. Laboratory L-alanine and DL-leucine were calibrated by elemental analysis/isotope ratio mass spectrometry (EA/IRMS) with reference to IAEA CH-6 (d13C ¼ –10.4%) and IAEA CH-7 (d13C ¼ –31.8%) to assess the accuracy of d13C measurements in amino acids by LC/IRMS. All solutions were prepared in freshly drawn water purified by reverse osmosis (18.2 MV.cm; Direct-QTM, Millipore, Watford, UK). LC/IRMS system The LC/IRMS system consisted of a Dionex ICS3000 ion chromatography system (Dionex, Sunnyvale, CA. USA) coupled through an interface (Liquiface, Isoprime Ltd., Cheadle Hulme, UK) to an isotope ratio mass spectrometer (IRMSr; Isoprime Ltd.).[2] It was operated in its standard configuration and in direct injection mode (DI-IRMS) where samples were injected directly into the interface via a six-port, three-way injection valve. Chromatography system set-up and conditions
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The LC system was operated under standard conditions (with pulsed amperometric detector (PAD)) except that a continuously regenerated anion trap column (CR-ATC) and degasser were plumbed in series between the pump and injector valve to limit carbonate deposition on the column. Both the CR-ATC and the degasser are proprietary Dionex parts. Underivatised amino acids were resolved with a strong
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anion exchange column (2 250 mm, AminoPac PA10 with a 2 50 mm AminoPac guard column; Dionex, Camberley, UK). In SAX chromatography, basic and neutral amino acids have been eluted and adequately resolved by dilute alkaline eluents but strong alkaline eluent coupled with the introduction of a counterion has been used to elute aromatic and acidic amino acids.[10,11] Increasing alkaline eluent strength potentially increases background CO2 and the recommended counterion for amino acid separation was acetate. Carbon-containing eluents are inappropriate in this application and other weakly bound inorganic ions have been used as counterions.[14] Several inorganic reagents were therefore tested as counterions at various concentrations; (2–60 mM) and NO Cl (2–50 mM), HPO2 4 3 (2–65 mM). Sodium nitrate was found to be most appropriate for this application (data not shown) since it introduced fewest baseline perturbations on the PAD and IRMSr, resulted in fewer extraneous peaks, had the shortest regeneration and equilibration time, and produced least perturbations to the CO2 background in the IRMS system. A similar counterion performance for the elution of sugars was recently reported.[9] The column temperature was held isothermally at 308C. When preparing eluents for SAX chromatography, background inorganic carbon (C)content (mainly from dissolved inorganic C after exposure of eluents to air) was minimised through careful reagent preparation using freshly drawn water by reverse osmosis and helium degassing of alkaline reagents. The amino acids were resolved on the LC system using a step-wise gradient of eluents beginning with an isocratic period (35 mM NaOH, 0.16 mL/min, 30 min). The hydroxide concentration was then increased to 180 mM in a three-step, stepped gradient (80, 120, 180 mM) at 3-min intervals between 30 and 36 min (0.23 mL/min). The increase in flow rate was to reduce the total run length. The flow rate was then decreased to 0.18 mL/min during the period 40–52 min. At 52 min, sodium nitrate was introduced into the mobile phase at 25 mM and held for 3 min, followed by a linear gradient decrease to 5 mM. These conditions were then maintained until the end of the run at 70 min. Chromatographic commands, data collection and handling were performed by ChromeleonTM software (Dionex, Sunnyvale, CA, USA).
Column regeneration The column was regenerated with 260 mM NaOH (0.25 mL/ min, 15 min) followed by an equilibration period (35 mM NaOH, 0.15 mL/min, 25 min). For the analysis of basic and neutral amino acids only, it was observed that after analysis of 16 samples in isocratic mode (35 mM NaOH), only 10-min regeneration (260 mM, 0.25 mL/min) and 10-min equilibration period were required for each batch. Once every month of continuous use, or after 100 samples, it was necessary to ’deep clean’ the column by running clean water (0.30 mL/min, 10 min) followed by dilute acid (145 mM HNO3, 0.25 mL/min, 1 h) and a second water rinse (30 min). The column was then regenerated using 1 M NaOH (0.25 mL/min, 4 h). Finally, the system was stabilised (35 mM NaOH, 0.25 mL/min, 30 min).
Copyright ß 2011 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2011, 25, 429–435
Amino acid d13C by SAX LC/IRMS The oxidation interface – precision and accuracy and C conversion efficiency The interface was modified in-house to improve resolution, reduce background carbon and maintain stable background carbon in the IRMS instrument.[2] The reagents (sodium persulfate (20%w/v) and phosphoric acid (10%v/v)) were pumped at 130 mL/min in each channel to the mixing union. In direct injection (offline) mode a third channel of water is used to carry the sample to the mixing tee and in ’online’ mode this channel is replaced by the LC system eluent flow. The offline isotope accuracy, precision and were determined by direct injection of samples (0.1–2 mM) each of alanine, DL-leucine, arginine, lysine, cyclo-leucine, methionine, histidine, phenylyalanine, glutamine, glutamic acid, glucose and sucrose (IAEA-CH-6; 0.025–1 mM). The organic C oxidation efficiency was determined with the interface reactor temperature set at both 908C and 998C. Alanine, DL-leucine (0.5–1 mM) and sucrose (IAEA-CH-6; 0.25–1 mM) were used to assess the oxidation efficiency, using the carbon-corrected CO2 yield from sucrose as a reference point. The helium counter flow at the separator can be varied to achieve a flow rate from 2 to 20 mL/min at the open split. The flow rate was maintained at 3.15 mL/min to ensure constant and high sensitivity for CO2 analysis by IRMS. The liquid effluent flow rate from the interface in direct injection mode was 0.4 mL/min, being the sum of three equal reagent flows.
n ¼ 3) were determined by EA/IRMS on a Roboprep-CN coupled to a 20-20 isotope ratio mass spectrometer (both from Sercon, Crewe, UK). This analysis provided an additional check of isotopic accuracy. The total organic C content and the d13C of bulk (unhydrolysed) prawn and BSA samples were similarly determined. More than 95% of the total organic C content of both materials was expected to be derived from protein. Tissue sample preparation and hydrolysis BSA and prawns were chosen to determine the d13C of amino acids from whole tissue (as opposed to separate amino acids). For prawns, bulk frozen samples were freeze-dried overnight and milled to a fine consistency. BSA was supplied as a fine homogeneous powder. Samples of prawns and BSA (10 mg each) were then hydrolysed (6 M HCl, 5 mL; 1508C, 4 h) in the gas phase under N2, in a PTFE pressure vessel within a ParrTM digestion bomb (Moline, IL. USA). Upon cooling, the samples were re-suspended in water and the hydrolysate diluted to 500–750 ng/mL, filtered (0.20 mm) and then dispensed into sampling vials. Aliquots (sample loop 25 mL) were analysed by LC/IRMS. Cyclo-leucine was used as an internal standard. The bulk d13C values of the prawns and BSA were determined as described previously using aliquots of hydrolysates (100–150 ng/mL; sample loop 20 mL). Data analysis
IRMS Isotopic measurements were performed on an IRMSr instrument (Isoprime Ltd.) with an electron ionisation (EI) ion source, differential pumping, an electromagnet and three Faraday cup detectors. The IRMSr was operated at an accelerating voltage of 3.465 kV. The ion source pressure was 2.2 106 mbar, and ions were generated by EI at an electron energy of 70 eV at a trap current of 400 mA. The three Faraday cup detectors monitored the [CO2]þ ion currents for m/z 44, 45 and 46, simultaneously and continuously. Calibration was achieved by admitting three pulses of CO2 reference gas (Air Products, Crewe, UK) into the inlet of the IRMS instrument for approximately 30 s each at the beginning of each run. The reference gas was independently calibrated by dual inlet IRMS and by back calibration against the IAEA-CH-6 sucrose standard using the interface in direct injection mode. Data collection and handling was conducted using IonVantageTM software (Isoprime Ltd.). The 13C abundance was expressed in the standard d-notation: Rsample d C ð%Þ ¼ 1 1000 Rstd 13
where Rsample and Rstd are the 13C/12C ratios in the sample and standard relative to the internationally accepted standard (Vienna Pee Dee Belemnite; VPDB), respectively. EA/IRMS
Rapid Commun. Mass Spectrom. 2011, 25, 429–435
Reconstituted bulk d13 C ¼ ðd13 C1 fC1 Þ þ ðd13 C2 fC2 Þ þ . . . þ ðd13 Cn fCn Þ 13 where d13Cn ¼ calculated P analyte d C, fCn ¼ fractional carbon contribution and fC ¼ 1. All the d13C measurements are the average of five replicates for online measurements, 16 measurements for direct injection, 3 measurements for EA/IRMS and the standard deviation (SD; 1s; %) was taken to be the precision of analysis. The Bland-Altman approach was used to assess the agreement between the d13C values derived for LC/IRMS and those for EA/IRMS.[16]
RESULTS Efficiency of carbon conversion into CO2 in the Liquiface The conversion of sample carbon into CO2 was determined at two reactor temperature settings of 908C and 998C for alanine, leucine and IAEA CH6 sucrose. The sensitivity (CO2 peak area, peak area ratios) was significantly greater ( p 0.05) between the average peak areas of alanine, leucine, glucose and sucrose at equi-carbon concentrations of 144 ng to 1440 ng (Table 1). The extent of recovery of carbon did not appear to significantly influence the measured d13C values; however, all the analyses were performed at 998C.
Copyright ß 2011 John Wiley & Sons, Ltd.
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The total organic C content and d13C of the powdered/ granular form of samples and standards (0.5 mg C equivalent;
The reconstituted bulk d13C signal was derived from online LC/IRMS data by weighting the individual amino acid d13C for fractional carbon content[15] using the equation:
D. A. Abaye, D. J. Morrison and T. Preston Table 1. Influence of oxidation temperature on C conversion into CO2 defined as m/z 44 signal intensity at 908C m/z 44 signal intensity at 998C and on d13C (n ¼ 3 for each analyte) 908C
Ala Std Ala Std IAEA-CH6 IAEA-CH6 Leu Std Leu Std
998C
Area ratio
Area
d13C
mM
ng C
Peak area
d13C
SD
Peak area
d13C
SD
908C/ 998C
908C vs. 998C
908C vs. 998C
1.00 0.50 0.50 0.25 1.00 0.50
720 360 1440 720 1440 720
5.72E-08 2.93E-08 1.14E-07 5.68E-08 1.03E-07 5.26E-08
–21.40 –24.10 –9.05 –9.05 –20.93 –20.98
1.41 1.20 0.92 0.92 0.12 0.26
8.06E-08 4.14E-08 1.59E-07 7.77E-08 1.40E-07 7.70E-08
–21.37 –21.57 –9.73 –10.67 –21.37 –20.95
0.17 0.32 0.12 0.34 0.19 0.17
0.71 0.71 0.71 0.73 0.73 0.68
p
