Sample Preparation and Measurement of Cholesterol in Pig Bile Using SPE and GC/MS

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Sample Preparation and Measurement of Cholesterol in Pig Bile Using SPE and GC/MS

The insertion of biliary endoprostheses (or biliary stents) is an established method in gastroenterology to ensure the drainage of bile.1,2 Despites its advantages, however, a major limitation of this endoscopic method is the accumulation of deposits, resulting in complete closure that requires stent replacement.3 The parameters supporting incrustation formation and accumulation are still unclear.4

While the role of bacteria is well-understood, the influence of the design and stent material is not.5 A bile-flow model was developed for the testing of newly developed biliary stents under nearly physiological conditions. The model considers the physiological temperature, position, and pressure of the bile duct as well as the discontinuous volume flow of bile.6 In addition to investigating the composition of stent incrustations, determining the composition of the test material pig bile of the bile-flow model provides information that facilitates the study of the incrustation process.7-10 This article describes a GC/MS method that was developed for the determination of cholesterol in pig bile. The complex matrix of the bile requires sample cleanup by solid-phase extraction.

Materials and methods

Chemicals, standards, and samples

Standard cholesterol (≥99%) and internal standard 5-a-cholestane (≥97%) were purchased from Sigma-Aldrich (St. Louis, MO); the solvents methanol (HPLC-grade) and hexane (HPLC-grade) were from Carl Roth (Karlsruhe, Germany). The GC reagent N-trimethylsilylimidazole (TSIM) was purchased from Macherey-Nagel (Bethlehem, PA). Pooled bile from a number of pigs was stored at –18 °C (–0.4 °F) in plastic syringes (vol. 300 mL). One syringe with bile was thawed prior to sample preparation; the remaining bile was stored at 4 °C (39.2 °F) and used for a maximum of seven days.

Instrumentation and parameters

Solid-phase extraction (SPE) was performed using a vacuum manifold for 12 columns from Macherey-Nagel and Strata-XL-A SPE cartridges (100 µm/500 mg/3 mL) with a polymeric strong anion exchange phase from Phenomenex (Torrance, CA). The standard valves made of plastic were replaced with metal valves to increase stability and simplify cleanup after the SPE run (see Figure 1).

ImageFigure 1 – Vacuum manifold with metal valves and Strata-XL-A SPE cartridges (100 µm/500 mg/3 mL).

The measurement system consisted of a 7890A gas chromatograph and triple-quadrupole mass spectrometer (G7000A with G7010 upgrade) from Agilent Technologies (Santa Clara, CA). Calibration of cholesterol was created in the range 100 μg/L–10 mg/L. Determination of the cholesterol concentration using GC/MS was performed with the following parameters: Helium was used as mobile phase; the stationary phase was a DB-17MS column (30 m × 250 µm × 0.15 µm) from Agilent Technologies. The samples were injected with a volume of 2 μL, a split ratio of 10:1, and an inlet temperature of 250 °C. Column flow rate was 1.2 mL/min.

The heating program started with a temperature of 200 °C with a hold time of 2 min, followed by a temperature-time ramp of 10 °C/min to 270 °C (hold time 1 min) and a further temperature-time ramp of 20 °C/min to a final temperature of 295 °C (hold time 2 min). The mass spectrometer was used with the following temperatures: 270 °C transfer line, 230 °C ion source, 150 °C quadrupole. Data acquisition was performed in SCAN mode (MS1 scan) with a mass range of 50–500 m/z and a scan time of 200 msec. The TMS derivative of cholesterol was identified and quantified using the ions with the m/z values of 458.1 (quantifier) and 368.0, 329.1, and 128.9 (qualifiers), and 5-α-cholestane with the m/z values 357.0 (quantifier) and 216.9, 148.9, and 262.0 (qualifiers).

Data acquisition and data preprocessing were performed using MassHunter GC/MS Acquisition B.07.02, MassHunter Qualitative Analysis B.07.00, and MassHunter Quantitative Analysis for QQQ B.07.00 instrument software from Agilent Technologies. In-house-developed Analytical Data Evaluation (ADE) software was used for final data interpretation and statistics.11-15

Solid-phase extraction and derivatization

To reduce viscosity, the pig bile was diluted with ultrapure water (1:5, v/v). The SPE cartridges were conditioned with 5 mL methanol and equilibrated with 5 mL ultrapure water. After the sample load of 1 mL pig bile, two wash steps were performed, each with 5 mL ultrapure water. Subsequently, the cartridges were dried for 30 min. Elution was done with 5 mL hexane. In the next step, 100 µL of 5-a-cholestane (internal standard) were added to reach a concentration of 1 mg/L. Finally, 1 mL of the final solution was transferred into GC vials (vol. 2 mL).

The derivatization procedure was developed based on the method from Cao et al.16 For derivatization, 25 µL of the TSIM GC reagent were added to the sample solution. The GC vials were capped and heated at 55 °C (131 °F) for 30 min.

Results and discussion

A series of experiments were performed to validate the method. The following validation parameters were determined: repeatability, recovery rate, within-laboratory precision, measurement precision, limit of detection (LOD), and limit of quantification (LOQ). A further validation parameter was recovery rate to ensure the correct value of the measurement results. The GC/MS method was validated using cholesterol standards dissolved in hexane (conc. 1.5 mg/L).

For validation of the SPE method, certified reference material was required. No reference material was commercially available for the special matrix of pig bile. In-house spiking of pig bile with cholesterol was not possible since solid standard cholesterol is not soluble in bile. The aim of stent testing is to monitor the composition of incrustations inside the stent and determine the stability and relative variation of the cholesterol concentration to fulfill the test requirements. Validation of the extraction method was performed using pooled pig bile. All validation parameters (excluding recovery rate) were determined similarly to validation of the GC/MS method. Figure 2 shows the calibration and two chromatograms.

ImageFigure 2 – a) Cholesterol calibration, b) chromatogram of standard sample, and c) chromatogram of pig bile sample.

Validation of GC/MS method

Repeatability and recovery rate were determined with 25 standard samples made of cholesterol dissolved in hexane with a final concentration of 1.5 mg/L. The mean value of cholesterol was 1.43 mg/L with a standard deviation (STDEV) of 0.05 mg/L and a coefficient of variation (CV) of 3.22%. Recovery rates ranged from 90% to 102% with a mean value of 95%. Within-laboratory precision was determined with 10 standard samples prepared and measured on five different days. CV values ranged from 1.1% to 3.2%. Measurement precision was determined with one sample measured 10 times. The CV value was 1.7%. LOD and LOQ were determined with 10 blank samples and calculated according to the equations in Ref. 8. LOD was 6.34 µg/L and LOQ was 17.06 µg/L. Table 1 summarizes the results.

table 1

Validation of SPE procedure

Repeatability was determined with 24 pig bile samples made of the same sample pool. A uniform SPE procedure (e.g., similar flow rates on each cartridge) can be ensured for the simultaneous preparation of six samples. Therefore, the samples were prepared and measured on two days with six samples in the morning and six samples in the afternoon. The mean value of cholesterol was 113.59 mg/L with a STDEV of 9.20 mg/L and a CV value of 8.10%. Within-laboratory precision was determined with five pig bile samples from the same sample pool prepared on five different days. CV values ranged from 3.6% to 10.6%. Measurement precision was determined with one pig bile sample measured 10 times. The CV value was 4.08%. LOD and LOQ were determined with 10 blank samples and calculated according to the equations in Ref. 8. LOD was 54.36 µg/L and LOQ was 142.95 µg/L. Table 2 and Figure 3 show the results.

table 2

ImageFigure 3 – Validation results (repeatability, within-laboratory precision, and measurement precision).

Summary and outlook

Solid-phase extraction combined with derivatization and GC/MS measurement enable the determination of cholesterol in pig bile. The method was validated using both cholesterol standard solutions and real pig bile samples. Future work will involve automation of this complex and time-consuming process.

References

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  10. Fleischer, H.; Thurow, K. Automation Solutions for Analytical Measurements—Concepts and Applications, Wiley-VCH 2017, ISBN 3-527-34217-6.
  11. Fleischer, H.; Adam, M. et al. IEEE I2MTC 2015, 1602–7.
  12. Fleischer, H.; Adam, M. et al. IEEE CASE 2015, 758–63.
  13. Adam, M.; Fleischer, H. et al. IMEKO TC4 2016, 278–83.
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Heidi Fleischer, Ph.D., is university lecturer at the University of Rostock, Institute of Automation, Richard-Wagner-Strasse 31, 18119 Rostock, Germany; tel.: +49 (0) 381 498 7803; fax: +49 (0) 381 498 7802; e-mail: [email protected]; www.uni-rostock.de. Kinjal Ramani, M.Sc., is junior scientist, and Kerstin Thurow, Ph.D., is CEO of the Center for Life Science Automation—celisca, Rostock, Germany. The authors wish to thank the German Federal Ministry of Education and Research for supporting the funding program KMU-innovativ: Medizintechnik, contract number 13GW0082D. Further thanks go to Detlef Behrend, Ph.D., Mareike Warkentin, Ph.D., and Caroline Deutsch, M.Sc., for providing the pig bile, and to Jenny Bandomir, Ph.D., for contributing to the development of the GC/MS method. Special thanks are expressed to Sybille Horn and Anne Reichelt for their excellent laboratory work.

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