Extraction of chloroquine and hydroxychloroquine from different biological matrices

For research use only. NOT for use in diagnostic procedures.

Figure 1. Structures of Chloroquine (CQ) and Hydroxychloroquine (HCQ).

Introduction

The objective of this study was to develop a fast and reliable extraction for Chloroquine (CQ) and Hydroxychloroquine (HCQ) from different human matrices (whole blood, serum and urine) using ISOLUTE® SLE+ Supported Liquid Extraction plates prior to LC-MS/MS analysis.

The simple sample preparation procedure delivers clean extracts and analyte recovery greater than 90% for CQ and greater than 80% for HCQ with minimal matrix effects and high process efficiency. 

Analytes

Chloroquine (CQ), and Hydroxychloroquine (HCQ)
Figure 2. Typical ISOLUTE® SLE+ procedure.

Sample preparation procedure¹

Format

ISOLUTE® SLE+ 400 µL Supported Liquid Extraction Plate (P/N 820-0400-P01)

Sample pre-treatment

Dilute the biological sample (100 µL) with 0.5M ammonium hydroxide (300 µL). Vortex briefly and let stand for 5 mins.

Sample loading

Load pre-treated samples (375 μL) onto the ISOLUTE® SLE+ 400 µL plate. Using a Biotage® PRESSURE+96 Positive Pressure Manifold (P/N PPM-96), apply a pulse of pressure to load samples onto the sorbent. Wait 5 minutes for the sample to equilibrate on the sorbent.

Elution

Apply ethyl acetate (750 μL) to each well, and allow to flow by gravity for 5 minutes. Apply a second aliquot of ethyl acetate (750 μL). Wait 5 minutes. For complete solvent recovery apply a pulse of positive pressure at 10 psi (10–20 seconds).

Evaporation

Dry the extract under a stream of nitrogen using a Biotage® SPE Dry at 40 °C, 20 to 40 L/min for approximately 25 minutes.

Reconstitution

Reconstitute with mobile phase A (100 µL) and mix thoroughly.

UHPLC conditions

Instrument

Agilent 1260 Infinity II LC System

Column

Restek Ultra AQ C18 3.0 um 100 X 2.1 mm. Cat # 9178312

Flow rate

0.4 mL/min

Column temperature

25 °C

Injection volume

5 µL

HPLC gradient

Table 1. HPLC gradient.

MS Sciex 4000 QQQ

Table 2 Mass spec source parameters.

Table 3 Transitions and retention times for target analytes in positive mode.

Results and discussion

Figure 3 shows an extracted ion chromatogram of a 10 ng/mL mixed standard solution of chloroquine and hydroxychloroquine. Two transitions for each analyte were used and total chromatographic separation was achieved.

Figure 4 shows an extracted ion chromatogram of a 10 ng/mL mixed solution of chloroquine and hydroxychloroquine spiked into blank human whole blood and extracted using the method described.
Figure 3. TIC and XIC for unextracted HCQ and CQ @10 ng/mL.
Figure 4. TIC and XIC for HCQ and CQ @10 ng/mL extracted from whole blood.

Figure 5 shows an extracted ion chromatogram of a 10 ng/mL mixed solution of chloroquine and hydroxy- chloroquine spiked into blank human serum and extracted using the method described.Figure 5. TIC and XIC for HCQ and CQ @10 ng/mL extracted from serum.

Figure 6 shows an extracted ion chromatogram of a 10 ng/mL mixed solution of chloroquine and hydroxychloro- quine spiked into blank human urine and extracted using the method described.
Figure 6. TIC and XIC for HCQ and CQ @10 ng/mL extracted from urine.

Matrix effects, analyte recovery and process efficiency were determined. Figures 7, 8, and 9 display matrix effects, recovery, and process efficiency respectively, obtained for chloroquine and hydroxychloroquine using the ISOLUTE® SLE+ 400 µL extraction plates. The study represents 3 sets of samples.²
Figure 7. Matrix effect.Figure 8. Recovery.Figure 9. Process effect.

Conclusion

ISOLUTE® SLE+ provides the selectivity and sensitivity required to extract and quantitate chloroquine (CQ) and hydroxychloroquine (HCQ) in a variety of biological fluid samples. The combination of a simple unified extraction for different matrices and the short run time will maximize the sample throughput and allow implementation of methods in high-throughput laboratories.

Additional information

• It is recommended to maintain column temperature at 25 ˚C with a 400 µL/min flow rate to allow the analytes to elute later in the gradient
• Carryover can be observed at higher concentrations. To reduce this:
• It is recommended to make mobile phase A as a mixture of aqueous and organic
• It is recommended to use a needle wash mix of acetonitrile, methanol, isopropanol and water (1:1:1:1, v/v) and smaller volume injections 2–5 μL
• Extending the column rinse step with 98% organic for 1 min and a re-equilibration time of 1 min. will also help reduce carryover
• The assay can be automated using the Biotage® Extrahera™
• When using deuterated internal standard, it should be prepared in 0.5 M ammonium hydroxide diluent

Ordering information

Chemicals and reagents

• Chloroquine diphosphate salt, hydroxychloroquine sulfate, methanol (LC-MS grade), acetonitrile (LC-MS grade), ammonium formate, water (LC-MS grade), formic acid (LC-MS grade), ethyl acetate (anhydrous, 99.8%), ammonium hydroxide were purchased from Sigma Aldrich (St. Louis, MO).
• Blank matrices (human whole blood, serum, and urine were purchased from UTAK labs (Valencia, CA)
• Mobile Phase A: ACN:20 mM Ammonium formate (15:85, v/v) + 0.2% formic was prepared in a 1 L bottle by combining 1.26 g of ammonium formate +150 mL of Acetonitrile + 850 mL of water + 2 mL of formic acid and mixed well, pH approximately 2.6
• Mobile Phase B: Methanol: ACN (75:25, v/v) was prepared in a 1 L bottle by combining 750 mL of methanol and 250 mL of acetonitrile and mixed well.
• 0.5M ammonium hydroxide was prepared by dissolving 1.75 g in 100 mL of water and mixed well.

References

1. Kaewkhao, Karnrawee, et al. “High Sensitivity Methods to Quantify Chloroquine and Its Metabolite in Human Blood Samples Using LC–MS/MS.” Bioanalysis, vol. 11, no. 5, 2019, pp. 333–347., doi:10.4155/bio-2018-0202.
2. Silvestro, Luigi, et al. “Matrix Effects in Mass Spectrometry Combined with Separation Methods - Comparison HPLC, GC and Discussion on Methods to Control These Effects.” IntechOpen, IntechOpen, 29 May 2013, www.intechopen. com/books/tandem-mass-spectrometry-molecular-characterization/matrix-effects-in-mass-spectrometry-combined-with-separation-methods-comparison-hplc-gc-and-discussi.
   
   

Literature Number: AN936