Page 54 - Pharmaceutical- Guide to Biopharmaceutical

Pharmacokinetics

Biomarker Discovery by Volatile Gas Analysis HS-20 Trap / GCMS-QP2020 NX

HS-20 / Nexis GC-2030 / SCD-2030

Analysis of Volatile Gases Generated

by Intestinal Microorganisms Cell Line Optimization
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Operating Principle and Features GC/MS (Comprehensive Analysis of Volatile Substances) • The HS-20 headspace sampler enables high-sensitivity analysis of volatile gases without pretreatment.
GC/MS (Comprehensive Analysis of Volatile Substances)
GC/MS (Comprehensive Analysis of Volatile Substances) • Using the HS-20 trap mode enables even higher sensitivity.
Microorganisms (flora) in intestines generate a wide variety of
volatile substances. Comprehensive analysis of such flora is used • The GC-SCD system enables selectively analyzing sulfur components with high sensitivity.
for biomarker discovery and other research. Volatile substances Culture
can be comprehensively analyzed using a gas chromatograph mass
spectrometer in combination with a headspace sampler unit (HS-20 +
GCMS-QP2020 NX).
Volatile sulfur compounds can be analyzed with high sensitivity by
using the headspace sampler in combination with an SCD detector that
detects only sulfur components with high sensitivity (HS-20 + Nexis Ex-GF mouse
Ex-GF mouse
GC-2030 + SCD-2030). GF mouse
Ex-GF mouse
10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 GF mouse
60.0
10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 55.0 60.0
GF mouse
Measurement Method and Conditions 10.0 15.0 20.0 Fig. 1 GC/MS Total Ion Chromatogram 50.0 55.0 60.0
45.0
30.0
40.0
35.0
25.0
Purification
GC-SCD (Selective Analysis of Sulfur-Based Volatile Substances) Specifications
Fresh fecal samples from both germ-free and flora-intact mice were GC-SCD (Selective Analysis of Sulfur-Based Volatile Substances)
placed directly into headspace vials. The vials were filled with anaerobic GC-SCD (Selective Analysis of Sulfur-Based Volatile Substances) Instrument HS-20 Trap / GCMS-QP2020 NX
gas, sealed, and left to cultivate for 24 hours. Then the vials were
placed in the headspace sampler and the evolved gases were analyzed HS unit Sample injection methods: Sample loop or adsorbent trap
Samples: 90
by GC/MS and GC-SCD. (For detailed analytical conditions, refer to the Oven temperature: Room temperature + 10 to 300 °C (1 °C steps)
site linked to the title.) Weight: 40 kg (HS-20Trap)
GC unit Oven temperature: Room temperature + 2 to 450 °C
Ex-GF mouse
Results Ex-GF mouse Carrier gas control: Constant liner velocity, constant pressure, or constant flowrate
Ex-GF mouse Flow controller pressure: Max. 970 kPa
The total ion chromatogram (TIC) from a comprehensive analysis of
GF mouse MS unit Ionization: EI (standard), CI, NCI (optional) Characterization
volatile substances (Fig. 1) and the chromatogram from analyzing GF mouse Mass range: m/z 1.5 to 1090
sulfur-based volatile substances (Fig. 2) both confirmed that a larger 0.0 0.0 5.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 GF mouse min Measurement modes: Scan, SIM, and scan/SIM simultaneous measurement
10.0
15.0
45.0
40.0
min
50.0
25.0
20.0
35.0
30.0
number and quantity of volatile substances were detected from the 0.0 5.0 10.0 15.0 Fig. 2 GC-SCD Chromatogram 40.0 45.0 50.0 min High-speed scan rate: 20,000 u/sec
30.0
25.0
35.0
20.0
flora-intact mouse than the germ-free mouse. They also confirmed that Weight: 85 kg for GC-MS main units and 10 kg for auxiliary pump
GC-SCD analysis can detect and identify sulfur compounds, which can Mouse C
have low peak intensity or overlap with other peaks in GC/MS results. 10 Mouse C
EX-GF3-1
10
EX-GF3-1
GC/MS data for 121 types of compounds acquired from two samples 10 Mouse C
taken from each of six mice with intact flora was analyzed by principal 5 Mouse E EX-GF3-2 EX-GF3-1
5 Mouse E EX-GF3-2
component analysis using SIMCA15 multivariate analysis software. Mouse F EX-GF7-2
EX-GF7-2
5 Mouse F Mouse E EX-GF3-2
In the score plot obtained from the above measurements (Fig. 3), the t[2] 0 Mouse F EX-GF8-2 EX-GF7-2 Mouse B Quality Control
EX-GF8-1
EX-GF7-1
EX-GF8-1
Mouse B
EX-GF7-1
smallest clusters were formed from samples taken from the same t[2] t[2] 0 EX-GF8-1 EX-GF8-2 EX-GF5-2 EX-GF7-1 EX-GF2-1 EX-GF2-2
EX-GF2-2
Mouse B
EX-GF5-1
individual. The individual-specific clusters clearly show that the system 0 EX-GF8-2 EX-GF5-2 EX-GF2-1 EX-GF2-2 EX-GF1-1
EX-GF1-1
EX-GF5-1
-5 EX-GF2-1 EX-GF1-2 Mouse A
can detect differences between individuals. -5 EX-GF5-2 Mouse D EX-GF1-2 EX-GF1-1
Mouse A
EX-GF5-1
Mouse D
-5 EX-GF1-2 Mouse A
Mouse D
-10
Conclusion -10
-10 Specifications
This example confirmed that volatile gases generated from intestinal -15 -15 -25 -20 -15 -10 -5 0 5 10 15 20
-10
10
-5
0
5
t[1]
flora can be analyzed without pretreatment using GC/MS and GC-SCD. -15 -25 -20 -15 R2X[1] = 0.54, R2X[2] = 0.141, Ellipse: Hotelling'sT2 (95 %) 15 20 Instrument HS-20/Nexis GC-2030 (SCD-2030 detector)
t[1]
SIMCA 15.0.2 -2020/04/21 13:48:33 (UTC+9)
-5
0
-10
10
5
-25 -20 -15 R2X[1] = 0.54, R2X[2] = 0.141, Ellipse: Hotelling'sT2 (95 %) 15 20
GC/MS enables comprehensive analysis, whereas GC-SCD enables t[1] SIMCA 15.0.2 -2020/04/21 13:48:33 (UTC+9) HS unit Sample injection method: Sample loop method
R2X[1] = 0.54, R2X[2] = 0.141, Ellipse: Hotelling'sT2 (95 %)
analysis of low-concentration sulfur components that are difficult to Fig. 3 Results (Score Plot) from Multivariate Analysis of Flora-Intact Mouse Analysis Results Samples: 90 Pharmacokinetics
SIMCA 15.0.2 -2020/04/21 13:48:33 (UTC+9)
detect by GC/MS. Using the methods in combination can be useful for Oven temperature: Room temperature + 10 to 300 °C (1 °C steps)
biomarker discovery and other pharmacokinetic applications. Weight: 33 kg (HS-20)
GC unit Oven temperature: Room temperature + 2 to 450 °C
We would like to acknowledge Principal Investigator Dr. Mitsuharu Matsumoto at Kyodo Milk
Application Examples Industry Co., Ltd. for providing samples and other help with conducting the above measurements. Sample injection unit temperature: Max. 450 °C
Carrier gas control: Constant liner velocity, constant pressure, or constant flowrate
• Comprehensive analysis of volatile components Flow controller pressure: Max. 970 kPa
Weight: 43.5 kg (SPL/FID model)
• High sensitivity analysis of volatile sulfur compounds Others
• Discovery of new biomarkers SCD unit Minimum detection quantity: <0.3 pgS/s (dibenzothiophene)
Dynamic range: >10 4
7
Selectivity: >10 (S/C)
Weight: 27 kg
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