Application Handbook Clinical Clinical The analysis of whole blood, plasma, serum and urine is a most Clinical applications benefit from Shimadzu’s complete port- insightful method in clinical research. Since the sensitivity folio covering chromatography and mass spectrometry of analytical instrumentations systems has improved steadily (GC, GC-MS, GC-MS/MS, HPLC, UHPLC, LC-MS, LC-MS/MS), in recent years, research results and reliability have increased spectroscopy (UV-Vis, FTIR, AAS, EDX, ICP-OES), life as well. sciences (MALDI-(TOF)-MS), microchip-electrophoresis, biopharmaceutical (aggregate sizer) and the observation In clinical applications, analytical instruments unfold a of medical microbubbles in targeted drug delivery using multitude of benefits: the HPV-X2 ultra high-speed camera. • They support the quality of human life. The concentration Shimadzu breaks new grounds by rethinking the use of of medications in Therapeutic Drug Monitoring (TDM), is mature technologies to develop new unique systems such assured, even though this may change according to age as the iMScope TRIO. It combines an optical microscope and health conditions and is dependent on gender, with a mass spectrometer for insights on the molecular level. genetic constitution or interferences with other drugs. For next-generation brain science, Shimadzu provides LABNIRS, an imaging technology for visualization of brain • They help to save lives, particularly when it comes to functions by functional near-infrared spectroscopy (fNIRS). time-critical situations, e.g. through acute intoxication, medical or drug abuse. • They analyze over- and undersupply of vitamins, minerals and trace elements. • They are applied in genomics, proteomics and metabolomics. • They uncover fraud in sports, particularly in animal or human doping. At the same time, analytical systems support health For Research Use Only. Not for use in diagnostic procedures. Not available in protection of animals and humans, even in the long-term. USA, Canada and China. Contents 1. Chromatography 1.1 Gas Chromatography 1.2 Liquid Chromatography 2. Mass Spectrometry 2.1 Gas Chromatography-Mass Spectrometry 2.2 Liquid Chromatography-Mass Spectrometry 3. Spectroscopy 3.1 Atomic Spectroscopy 3.1.1 Atomic Absorption Spectroscopy (AAS) 3.1.2 Energy Dispersive X-Ray Fluorescence (EDX) 3.1.3 Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) 3.2 Molecular Spectroscopy 3.2.1 Ultraviolet Visible Spectroscopy and Near Infrared (UV-VIS NIR) 3.2.2 Fourier Transform Infrared Spectroscopy (FTIR) 3.2.3 Fluorescence Spectroscopy 4. Life Science Lab Instruments 4.1 Matrix Assisted Laser Desorption Ionization (MALDI) 1. Chromatography 1. Chromatography 1.1 Gas Chromatography Chromatographic separation in gas phase for analysis of LAAN-J-GC-E012 Analysis of alcohol compounds in volatile and semi volatile components is used in clinical field blood (1) since many years. Gas chromatography is a key technique for LAAN-J-GC-E013 Analysis of alcohol compounds in quantitative analysis of alcohol in blood. blood (2) LAAN-J-GC-E012 GC Gas Chromatography Analysis of Alcohol Compounds in Blood (1) 12 Measurements of oxygenated compounds and alcohols, primarily ethanol, in blood, are frequently performed in forensic medicine, emergency medicine, and other fields. In forensic medicine, such measurements are utilized to determine levels of intoxication from alcohol consumption and to evaluate criminality. In emergency medicine, they are utilized to distinguish between alcohol consumption and other medical cases. For such analyses, systems equipped with a headspace sampler and GC-FID detector are often used. These measurements must be convenient and quick, and provide high-accuracy results. The Shimadzu HS-20 headspace sampler is capable of meeting these demands. When the HS-20, GC-2010 Plus, and a workstation (LabSolutions LC/GC) are used in combination, everything including the headspace conditions and GC conditions can be controlled from the workstation, which makes it easy to configure settings and operate the system. This report examines the repeatability and linearity of a standard ethanol solution, and confirms the separation of other oxygenated compound standard solutions. The analysis presented here was performed using the HS-20 and the Rtx-BACPlus series of high-separation columns specifically designed for the analysis of alcohol in blood. Analysis Conditions HS-20 Shared Conditions Oven Temp.: 85 C Vial Agitation: Off Vial Warming Time: 15 min. Vial Pressurization: 100 kPa Vial Pressurization Time: 1 min. Load Time: 0.5 min. Injection Time: 0.5 min. Needle Flash Time: 0.5 min. Sample Line Temp.: 150 C Transfer Line Temp.: 150 C Vial Volume: 20 mL GC-2010 Plus AF + LabSolutions LC/GC Columns: (1) Rtx-BAC Plus 2, 0.32 mm 30 m, d.f. 0.6 m (for screening) (2) Rtx-BAC Plus 1, 0.32 mm 30 m, d.f. 1.8 m (for separation confirmation) Column Temp.: 40 C Carrier Gas Pressure: 100 kPa (helium pressure mode) Split Ratio: 1:20 FID Temp.: 250 C Hydrogen: 40 mL/min. Makeup Gas: 30 mL/min. (helium) Air: 400 mL/min. Results Table 1 shows area value repeatability for standard solutions (0.1 mg/mL) of ethanol and t-butanol, and area ratio repeatability for ethanol and t-butanol, both obtained using the Rtx-BAC Plus 2 column. Fig. 1 shows overlapping chromatograms for ethanol in the standard solution. As indicated, favorable repeatability was obtained, with an ethanol area value repeatability of 1.42 %, and area ratio repeatability of 0.62 %. Fig. 2 shows the linearity of ethanol in the 0.1 mg/mL to 1.6 mg/mL range. It is evident that favorable linearity was obtained, with R = 0.9999 or higher. Fig. 3 shows an example of the separation of a standard solution with seven oxygenated components, including alcohol, using the Rtx-BAC Plus 2. All peaks were successfully separated within 3 minutes. Fig. 4 shows the separation of a standard solution with seven oxygenated components using the Rtx-BAC Plus 1. The separation pattern shown is different from that with the Rtx- BAC Plus 2, so qualitative ability is increased by performing analysis with both columns. Fig. 5 shows the separation of a standard solution with 10 oxygenated components using the Rtx-BAC Plus 1. The 10 oxygenated components were successfully separated in 5 minutes. Table 1 Repeatability (n = 7) of Peak Area Values for 0.1 mg/mL of Ethanol and t-Butanol 1234567meanRSD% Ethanol 57886 58682 57373 57753 58193 56139 57141 57595 1.422 t-Butanol 969772 980873 964274 958311 969829 949515 953278 963693 1.128 Ethanol/ 0.05969 0.059826 0.059499 0.060265 0.060003 0.059124 0.059942 0.059764 0.622 t-Butanol Table 2 Repeatability (n = 7) of Retention Times for 0.1 mg/mL of Ethanol and t-Butanol 1234567meanRSD% Ethanol 1.691 1.691 1.691 1.692 1.691 1.692 1.691 1.691 0.0230 t-Butanol 2.117 2.117 2.117 2.118 2.117 2.118 2.117 2.117 0.0180 12 Conc. (100) Ethanol External standard method Calibration curve: Straight line Through the origin: No Weighting: None Mean RF: 1.674826e-006 RF standard deviation: 1.019928e-007 1.55 1.60 1.65 1.70 1.75 1.80 1.85 min Area RF relative standard deviation: 6.089755 Fig. 1 Repeatability (n = 7) for a 0.1 mg/mL Fig. 2 Linearity of 0.1 mg/mL to 1.6 mg/mL Standard Solution of Ethanol Standard Solutions of Ethanol 4 67 1 3 1 Acetaldehyde 5 2 Methanol 3 Ethanol 4 Acetone 2 5 Isopropanol 6 t-Butanol 7 1-Propanol 0.5 1.0 1.5 2.0 2.5 3.0 min Fig. 3 Chromatograms for Seven Components (0.1 mg/mL each) Obtained Using the Rtx-BAC Plus 2 46 1 1 Acetaldehyde 2 Methanol 7 3 Ethanol 3 4 Acetone 5 Isopropanol 5 6 t-Butanol 7 1-Propanol 2 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min Fig. 4 Chromatograms for Seven Components (0.1 mg/mL each) Obtained Using the Rtx-BAC Plus 1 1 46 9 1 Acetaldehyde 8 2 Methanol 5 3 Ethanol 3 7 4 Acetone 5 Isopropanol 10 6 t-Butanol 7 1-Propanol 8 Acetonitrile 2 9 Methylethylketone 10 Ethyl acetate 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min Fig. 5 Chromatograms for 10 Components (0.1 mg/mL each) Obtained Using the Rtx-BAC Plus 1 First Edition: October 2013 Shimadzu Corporation www.shimadzu.com/an/ Shimadzu Corporation, 2013 LAAN-J-GC-E013 GC Gas Chromatography Analysis of Alcohol Compounds in Blood (2) 13 Measurements of oxygenated compounds and alcohols, primarily ethanol, in blood, are frequently performed in forensic medicine, emergency medicine, and other fields. In forensic medicine, such measurements are utilized to determine levels of intoxication from alcohol consumption and to evaluate criminality. In emergency medicine, they are utilized to distinguish between alcohol consumption and other medical cases. Application Data Sheet No. 12 introduced results for the repeatability of ethanol and the separation of standard solutions of oxygenated compounds using the HS-20 and the Rtx-BAC Plus series of columns specifically designed for alcohol analysis. This report introduces the results of an investigation of linearity and repeatability for blood spiked with ethanol. Analysis Conditions HS-20 Shared Conditions Oven Temp.: 85 C Vial Agitation: Off Vial Warming Time: 15 min. Vial Pressurization: 100 kPa Vial Pressurization Time: 1 min. Load Time: 0.5 min. Injection Time: 0.5 min. Needle Flash Time: 0.5 min. Sample Line Temp.: 150 C Transfer Line Temp.: 150 C Vial Volume: 20 mL GC-2010 Plus AF + LabSolutions LC/GC Column: Rtx-BAC Plus 2, 0.32 mm 30 m, d.f.