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User Benefits Application News A new quick and simple pretreatment allows for the measurement of ethylene glycol levels in blood by simultaneously removing proteins and derivatization. Since it uses the same mobile phase as the “LC/MS/MS Rapid Toxicology Screening System”, it can be run on the same system with just a column change. This method does not use expensive reagents and isotope-labeled internal standards, slowing for low-cost running. LC-MS/MS Analysis of Ethylene Glycol in Blood Using a Simple Derivatization Method Toshinari Ishii High Performance Liquid Chromatograph Mass Spectrometer System : Nexera XR + LCMS-8050 RX Column : Shim-pack Velox HILIC (Shimadzu, 2.1 mm i.d. 100 mm length, 2.7 µm) Column Temp. : 30 °C Injection Volume : 1 µL Rinse Setting : Sample interior/exterior cleaning Rinse Solution : MeOH Mobile Phase : A) 10 mM ammonium formate + 0.1 % formic acid (in water) : B) 10 mM ammonium formate + 0.1 % formic acid (in MeOH) Flowrate : 0.3 mL/min Time Program (%B) : 90 % (0 - 1.5 min)→20 % (1.5 - 4.0 min) → 90 % (4.0 - 7.0 min) Nebulizing Gas : 2 L/min Drying Gas : 10 L/min Heating Gas : 10 L/min DL Temp. : 150 °C Heat Block Temp. : 200 °C Interface Temp. : 300 °C Compound Name Quantitative Ion Reference Ion EG-BPBA 228.0 > 148.0 (CE -20 V) 230.0 > 148.0 (CE -20 V) PD-BPBA (Internal Standard) 270.0 > 184.0 (CE -15 V) 272.0 > 186.0 (CE -15 V) Table 1 Analysis Conditions MRM Transitions Analysis Conditions Analysis was performed using an LCMS-8050 RX (Fig. 3) using the conditions shown in Table 1. The derivative EG-BPBA contains bromine and is detected as a characteristic isotope ion. The MRM transitions below were chosen because the EG-BPBA isotope ions (m/z 228 and 230) are detected with about the same intensity, so they can be used for identification. Fig. 3 Nexera with LCMS-8050 RX Introduction Ethylene glycol is an industrial product used in antifreeze and other applications. However, it is also a toxic substance that causes metabolic acidosis and renal failure when absorbed by the body. As a highly polar, low molecular weight molecule, ethylene glycol is only weakly retained on reversed-phase columns and is not readily detected by LC-MS analysis without some form of pretreatment. It is also highly hydrophilic, difficult to isolate from blood, and not easily measured by GC without pretreatment. These issues led to the development of a GC-MS method that detects phenylboronic derivatives of ethylene glycol produced by derivatization reactions in aqueous solutions. However, the phenylboronic acid used by this method can accumulate in GC inserts and columns, affecting subsequent measurements. This study investigated using a boronic acid derivatization reagent (2-bromopyridine-5-boronic acid [BPBA]) compatible with LC-MS analysis to develop a simple and rapid method for measuring ethylene glycol levels in blood. BPBA selectively reacts with the diol groups of ethylene glycol, forming a stable cyclic boronic ester (Fig. 1). The nitrogen atom in this cyclic boronic ester improves its electrospray ionization (ESI) efficiency, whereas BPBA reacts quickly with ethylene glycol in aqueous samples. 1) Taking advantage of these attributes, a simple and rapid pretreatment process was developed that performs deproteinization and derivatization simultaneously. Fig. 1 Derivatization Reaction Sample Pretreatment The pretreatment workflow is shown in Fig. 2. Deproteinization and derivatization were performed simultaneously by adding 390 μL of 0.1 % TFA-acetonitrile containing 2.5 mM of BPBA to the sample and mixing it in a vortex mixer. The mixture was then centrifuged at 12,000 g for 5 minutes and the supernatant was recovered for analysis. 1,2-Pentanediol (100 µg/mL) was used as an internal standard. N Br B O O Fig. 2 Sample Pretreatment Workflow 100 µL of sample (reference solution or whole blood) 10 µL of internal standard added 390 µL of 0.1 % TFA-acetonitrile containing BPBA (2.5 mM) added Centrifugation (12,000 g, 5 min, 4 °C) Collect supernatant About 10 minutes N Br B OH HO Ethylene glycol BPBA EG-BPBA
Application News www.shimadzu.com/an/ Shimadzu Corporation © Shimadzu Corporation, 2025 For Research Use Only. Not for use in diagnostic procedures. This publication may contain references to products that are not available in your country. Please contact us to check the availability of these products in your country. The content of this publication shall not be reproduced, altered or sold for any commercial purpose without the written approval of Shimadzu. See http://www.shimadzu.com/about/trademarks/index.html for details. Third party trademarks and trade names may be used in this publication to refer to either the entities or their products/services, whether or not they are used with trademark symbol “TM” or “”. Shimadzu disclaims any proprietary interest in trademarks and trade names other than its own. The information contained herein is provided to you "as is" without warranty of any kind including without limitation warranties as to its accuracy or completeness. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication. This publication is based upon the information available to Shimadzu on or before the date of publication, and subject to change without notice. Nexera, and Shim-pack Velox are trademarks of Shimadzu Corporation or its affiliated companies in Japan and/or other countries. Concentration (µg/mL) 1st Analysis 2nd Analysis 3rd Analysis 4th Analysis 5th Analysis Accuracy % RSD% 98.6 92.2 95.7 97.1 98.4 96.4 2.4 228.0>148.0 (+) 230.0>148.0 (+) Table 2 Accuracy and repeatability of spiked EG in blood <References> 1) S Shen, F Zhang, S Zeng, J Zheng, “An approach based on liquid chromatography/electrospray ionization–mass spectrometry to detect diol metabolites as biomarkers of exposure to styrene and 1,3-butadiene,” Analytical Biochemistry, 386 (2009), p.186- 193 Conclusion This article describes a simple and quick sample pretreatment procedure for quantifying levels of ethylene glycol in blood. When phenylboronic derivatives are used to measure ethylene glycol by GC-MS, the derivatization reagent can accumulate in the equipment, affecting subsequent measurements. However, with the new method, the LC flow lines (including the autosampler) can be cleaned with solvents that have a high affinity for BPBA, which eliminates derivatization reagent carryover. The mobile phases used with this method are also used in Shimadzu’s method package products, such as the “LC/MS/MS Rapid Toxicology Screening System”. Therefore, with just a column change, toxicological drug screening and ethylene glycol measurements can both be performed with the same system. Y = 0.0365620X + 0.177183 R = 0.99996 0 50 100 150 200 250 300 350 400 450 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 500 PD-BPBA 0 0.5 1.0 1.5 0 1.0e4 2.0e4 3.0e4 4.0e4 270.0>184.0 (+) 272.0>186.0(+) Fig. 4 MRM Chromatograms for EG-BPBA (10 µg/mL) and PD-BPBA Fig. 5 Calibration Curve Fig. 6 High Concentration Ethylene Glycol Analysis after Multiple Successive Measurements Evaluation of Linearity and Sensitivity Reference samples of aqueous solutions of ethylene glycol were analyzed to verify the linearity of the method. Given that ethylene glycol is toxic at blood concentrations of 200 µg/mL and above, reference samples of 10, 20, 50, 100, 200, and 500 µg/mL were prepared for analysis. Fig. 4 shows chromatograms of EG-BPBA (10 µg/mL) and PD-BPBA. The reference sample results show that the method is sensitive enough to detect ethylene glycol at 1/20 of the level that is toxic. The calibration curve is shown in Fig. 5. The results showed good linearity (R = 0.99996). A solution of ethylene glycol (1000 µg/mL) without BPBA that was analyzed after verifying its linearity produced a chromatogram with no EG-BPBA peak (Fig. 6). This indicates that BPBA is unlikely to remain in equipment flow lines and affect subsequent analyses. Validity Assessment with Spiked Whole Blood Sample Ethylene glycol was added to whole blood to a concentration of 100 µg/mL and used to evaluate the repeatability and accuracy (n = 5) of the method. The results showed good accuracy (96.4 %) and repeatability (2.4 %) (Table 2). Analysis of a blank whole blood sample with no added ethylene glycol resulted in a peak with 1/20 the intensity of the 10 µg/mL reference solution (Fig. 7). 0.5 1.0 1.5 0 2.0e4 4.0e4 6.0e4 8.0e4 0 0.0 0.5 1.0 1.5 0.0e0 2.0e2 4.0e2 6.0e2 8.0e2 1.0e3 0.0 0.5 1.0 1.5 5.0e4 1.0e5 1.5e5 2.0e5 EG-BPBA 0.0 0.5 1.0 1.5 0.0e0 1.0e2 2.0e2 3.0e2 4.0e2 5.0e2 6.0e2 228.0>148.0 (+) 230.0>148.0 (+) EG-BPBA 228.0>148.0 (+) 230.0>148.0 (+) Fig. 7 MRM Chromatograms Showing EG-BPBA in Blank and Spiked Whole Blood (100 µg/mL) Sample Concentration (µg/mL) Relative Intensity 228.0>148.0 (+) 230.0>148.0 (+) Blank Spiked Whole Blood First Edition: Nov. 2025 01-01041-EN
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