Protocol for Therapeutic Drug Monitoring Within the Clinical Range Using Mid-infrared Spectroscopy

Abstract

Therapeutic drug monitoring (TDM), which involves measuring drug levels in patients' body fluids, is an important procedure in clinical practice. However, the analysis technique currently used, i.e. liquid chromatography-tandem mass spectrometry (LC-MS/MS), is laboratory-based, so does not offer the short response time that is often required by clinicians. We suggest that techniques based on Fourier transform infrared spectroscopy (FTIR) offer a promising alternative for TDM. FTIR is rapid, highly specific and can be miniaturized for near-patient applications. The challenge, however, is that FTIR for TDM is limited by the strong mid-IR absorption of endogenous serum constituents. Here, we address this issue and introduce a versatile approach for removing the background of serum lipids, proteins and small water-soluble substances. Using phenytoin, an antiepileptic drug, as an example, we show that our approach enables FTIR to precisely quantify drug molecules in human serum at clinically relevant levels (10 μg/mL), providing an efficient analysis method for TDM. Beyond mid-IR spectroscopy, our study is applicable to other drug sensing techniques that suffer from the large background of serum samples.

Figure 1

Two FTIR techniques, ATR and PM-IRRAS, to measure dried phenytoin spots. Schematic of the principles of ATR-FTIR (a) and PM-IRRAS (c); (b) ATR-FTIR spectrum measured from a dried spot of phenytoin dissolved in ethyl acetate at a concentration of 20 μg/mL (orange line) and a blank sample (blue line). The two peaks at 1772 and 1726 cm^–1 (highlighted by arrows) correspond to the asymmetric and symmetric stretching of the carbonyl group of the molecule, respectively; (d) PM-IRRAS spectrum measured from of a dried spot of phenytoin dissolved in ethyl acetate at a concentration of 5 μg/mL and a blank sample. The inset shows the chemical structure of phenytoin. The blue shadings in (b) and (d) represent the measurement noise calculated as the 3-fold standard deviation (3σ) of ten repeated measurements of blank samples. This noise primarily includes atmospheric noise from water vapor and carbon dioxide.

Figure 2

(a) PM-IRRAS spectra following protein removal by protein precipitation (dotted line) and liquid–liquid extraction (dash-dotted line), compared to dried serum proteins (dashed line) and pure phenytoin (solid line); The y-axis values are offset for clarity. (b) PM-IRRAS spectra of dried spots of phenytoin with and without serum lipids at concentrations of 10 and 50 μg/mL. With the presence of serum lipids (orange and blue lines), the 1772 cm^–1 target peak is significantly diminished.

Figure 3

Protocol for human serum sample preparation to remove lipids, small water-soluble substances and proteins and water. (a) Schematic diagram of the protocol. Step 1, lipid removal: a mixture of magnesium chloride (3 mol/L)-dextran sulfate sodium (6%) is added to precipitate lipoproteins, to which serum lipids such as triglycerides and cholesterol bind. Step 2, removal of small water-soluble substances: saturated ammonium sulfate is added to precipitate hydrophilic proteins such as albumin, to which the target drug phenytoin binds. The supernatant, involving small water-soluble molecules, is subsequently discarded. Step 3, protein removal: the water-immiscible solvent ethyl acetate is used to extract phenytoin while removing serum proteins. Step 4, dried spot: the upper solvent layer is collected and dried on a gold-coated silicon substrate for PM-IRRAS measurements; (b) PM-IRRAS spectra highlighting the successive reduction of the serum background after the various steps used in the protocol; (c) A zoomed-in spectrum of the 1772 cm^–1 peak highlighting the successful conclusion of the protocol. A concentration of 10 μg/mL is clearly identifiable, well above the 3σ of serum background, calculated from six replicates (indicated by blue shading).

Figure 4

Calibration curve of PM-IRRAS FTIR and validation of serum samples. (a) PM-IRRAS FTIR spectra of phenytoin spiked samples at concentrations of 10, 15, 20, 25, 30 μg/mL, along with blank serum, following preparation with our new protocol. Each spectrum is normalized to the peak height of the internal standard at 2221 cm^–1. Inset: a zoom-in spectrum of the target peak at 1772 cm^–1; (b) calibration curve of phenytoin spiked serum measured via PM-IRRAS. Six replicates are measured for each concentration; (c) validation measurements of PM-IRRAS FTIR compared to HPLC at two concentrations within the range shown in (b). Each concentration was analyzed in six replicates.