Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Summary

Current methods of analyzing patients’ adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

Abstract

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. Plasma drug levels are the current gold standard assessment, but require phlebotomy and a cold chain, and capture only very recent adherence. Our method uses hair, a matrix that is easily collected and reflective of long-term adherence, to test for 11 anti-TB medications. Previous work by our group shows that antiretroviral drug levels in hair are associated with HIV outcomes. Our method for DR-TB drugs uses 2 mg of hair (3 cm proximal to the root), which is pulverized and extracted in methanol. Samples are analyzed with a single LC-MS/MS method, quantifying 11 drugs in a 16 min run. Lower limits of quantification (LLOQs) for the 11 drugs range from 0.01 ng/mg to 1 ng/mg. Drug presence is confirmed by comparing ratios of two mass spectrometry transitions. Samples are quantified using the area ratio of the drug to the deuterated, ¹⁵N-, or ¹³C-labeled drug isotopologue. We used a calibration curve ranging from 0.001-100 ng/mg. Application of the method to a convenience sample of hair samples collected from DR-TB patients on directly observed therapy (DOT) indicated drug levels in hair within the linear dynamic range of nine of the eleven drugs (isoniazid, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, clofazimine, bedaquiline, pretomanid). No patient was on prothionamide, and the measured levels for ethionamide were close to its LLOQ (with further work instead examining the suitability of ethionamide’s metabolite for monitoring exposure). In summary, we describe the development of a multi-analyte panel for DR-TB drugs in hair as a technique for therapeutic drug monitoring during drug-resistant TB treatment.

Introduction

In the twenty-first century, drug-resistant TB (DR-TB) is an evolving catastrophe for already weak national TB control programs, with confirmed cases doubling in the past 5 years alone, accounting for nearly one-third of all deaths related to antimicrobial resistance globally^1,2. Successful treatment of DR-TB has conventionally required longer and more toxic second-line regimens than treatment for drug-sensitive TB. Moreover, patients with DR-TB often have significant pre-existing challenges to adherence, which contributed to the emergence of resistance initially³.

Unlike HIV infection where viral loads can be used to monitor treatment, surrogate endpoints of treatment response in TB are delayed and unreliable on an individual level⁴. Monitoring patient adherence, an important predictor of subtherapeutic anti-TB drug concentration and treatment failure, is also challenging. Self-reported adherence suffers from recall bias and the desire to please providers^5,6. Pill counts and medication event monitoring systems (MEMS) can be more objective⁷ but do not measure actual drug consumption^8,9,10. Drug levels in biomatrices can provide both adherence and pharmacokinetic data. Therefore, plasma drug levels are commonly used in therapeutic drug monitoring^11,12. In the context of drug adherence monitoring, however, plasma levels represent short-term exposure and are limited by significant intra- and inter-patient variability when determining appropriate adherence reference range. “White coat” effects, where adherence improves prior to clinic or study visits, further complicates the ability of plasma levels to provide accurate drug adherence patterns¹³.

Hair is an alternative biomatrix that can measure long-term drug exposure^14,15. Many drugs and endogenous metabolites incorporate into the hair protein matrix from the systemic circulation as hair grows. As this dynamic process continues during hair growth, the amount of drug deposited in the hair matrix depends on the continuous presence of the drug in circulation, making hair an excellent temporal readout of drug intake. Hair as a biomatrix has the additional advantage of being easily collected without the need for cold chain for storage and shipment compared to blood. Moreover, hair is non-biohazardous, which provides additional feasibility advantages in the field.

Hair drug levels have long been used in forensic applications¹⁶. Over the last decade, hair antiretroviral (ARV) levels have demonstrated utility in assessing drug adherence in HIV treatment and prevention, to which our group contributed. ARV levels in hair have been shown to be the strongest independent predictors of treatment outcomes in HIV infection^{17,18,19,20,21}. To determine whether hair levels of DR-TB patients will have the same utility in predicting treatment outcome, we used LC-MS/MS to develop and validate a method for analyzing 11 DR-TB medications in small hair samples. As an initial assessment of the assay’s performance, we measured DR-TB drugs levels in a convenience sample of patients with DR-TB receiving directly observed therapy (DOT) in the Western Cape, South Africa²².

Protocol

All patients provided written informed consent prior to hair sample collection. We obtained Institutional Review Board approval from the University of Cape Town and the University of California, San Francisco.

1. Hair sampling

1. Obtain written informed consent.
2. Use clean scissors to cut approximately 20-30 scalp hair strands from the occipital region as close to the scalp as possible.
3. Place tape around the distal side of the hair to indicate directionality. Fold hair sample into an aluminum foil square and store at room temperature. Label the distal end of the hair to avoid possible contamination from additional handling of the proximal end.
4. In addition to patient samples, collect “blank hair”: a scalp hair sample from someone who has not taken TB medication. Collect a large amount (>30 mg blank hair for each 20 patient samples).

2. Drug extraction

1. Label bead tubes. Each patient sample requires one tube. Label 12 tubes from “C0” to “C11”, one for each of the 12 calibration points. Label a tube for Low Quality Control, a tube for Medium Quality Control, and a tube for High Quality Control. Lastly, label a tube for Matrix Blank.
2. Open the aluminum foil square containing hair sample. If hair sample is longer than 3 cm, cut hair at 3 cm from the proximal end and use that proximal portion for analysis.
3. Weigh 2 mg of the hair sample into a bead tube.
4. Weigh 2 mg of blank hair into 16 additional bead tubes. These will be used as the calibration points, quality controls, and matrix blank. The tubes will follow the same extraction procedure as the patient samples, aside from being spiked with drug reference standards at levels indicated in steps 2.8 and 2.9.
5. Place all of the bead tubes into the homogenizer. Run homogenizer at speed 6.95 m/s. Run for two cycles of 30 s each, with a 15 s rest period in between the two cycles.
6. Make internal standard mix and add it to the samples.
   1. Add ~40 mL of methanol to a 50 mL volumetric flask.
   2. In amber glass vials, make the mixes of the internal standards shown in Table 1, using methanol as the solvent.
      NOTE: Methanol is very volatile. Leave all vials capped during this process to prevent loss due to evaporation.
   3. From those mixes, add the volume shown in Table 1 to the 50 mL volumetric flask. Then, fill the flask to 50 mL with methanol.
   4. Cap and mix the volumetric flask. Add 500 μL of the mixture to each of the pulverized hair tubes, except for the matrix blank tube. Add 500 μL methanol to the matrix blank tube.
7. Make reference standard mixes.
   1. Constitute neat reference standards of the following drugs with methanol to get the concentration shown in the table in step 2.7.2. Only 1 mL of the following concentrations is necessary.
   2. Add 1768 μL of methanol to a vial, and then add the amounts of reference standard listed in Table 2 to the same vial to get 2 mL final volume. Label this vial “Ref Std Mix 1x”. Vortex.
   3. Spike 100 μL from “Ref Std Mix 1x” into 900 μL methanol in a new vial. Label this vial “Ref Std Mix 10x”. Vortex.
   4. Spike 100 μL from “Ref Std Mix 10x” into 900 μL methanol in a new vial. Label this vial “Ref Std Mix 100x”. Vortex.
   5. Spike 100 μL from “Ref Std Mix 100x” into 900 μL methanol in a new vial. Label this vial “Ref Std Mix 1000x”. Vortex.
8. Spike the calibration curve tubes by adding the amount of Ref Std Mix described in Table 3.
9. Create QC mixes and spike quality control tubes.
   1. Label 5 vials “QC-A” through “QC-E”.
   2. Add the following amounts of methanol to the five labeled vials:
      QC-A: 990 μL
      QC-B: 940 μL
      QC-C: 950 μL
      QC-D: 980 μL
      QC-E: 950 μL
       
   3. Using the 1 mg/mL drug stocks created in step 2.7.1, add 10 μL to the specific vials listed below. For the BDQ and CLF stocks, which are at 0.5 mg/mL, add 20 μL to the vials listed below.
      QC-A: PTH
      QC-B: EMB, CLF, BDQ, PTM
      QC-C: INH, LFX, LZD, MFX, PZA
      QC-D: PTH, EMB
      QC-E: CLF, BDQ, PTM
      
      NOTE: Some drugs are present in multiple mixes.
   4. Label a vial as “QC-A df100”. Dilute 10 μL of QC-A into 990 μL methanol.
   5. Label a vial as “Low QC stock”. Add 1832 μL methanol to this vial. Add the amounts of QC mixes detailed below:
      QC-A df100: 80 μL
      QC-B: 8 μL
      QC-C: 80 μL
       
   6. Label a vial as “Mid QC stock”. Add 760 μL methanol to this vial. Add the amounts of QC mixes detailed below:
      QC-A df100: 800 μL
      QC-B: 40 μL
      QC-C: 400 μL
       
   7. Label a vial as “High QC stock”. Add 1376 μL methanol to this vial. Add the amounts of QC mixes detailed below:
      QC-D: 160 μL
      QC-E: 320 μL
      MFX, 1mg/mL stock: 16 μL
      INH, 1mg/mL stock: 32 μL
      LFX, 1mg/mL stock: 32 μL
      LZD, 1mg/mL stock: 32 μL
      PZA, 1mg/mL stock: 32 μL
       
   8. Spike 10 μL Low QC stock into the Low QC bead tube.
   9. Spike 10 μL Mid QC stock into the Mid QC bead tube.
   10. Spike 10 μL High QC stock into the High QC bead tube.
10. Place all tubes in hot shaker for 2 h at 37 °C. Shaking should be slow enough that the water does not splash up on to the tubes.
11. Remove tubes from shaker. Transfer liquid from bead tubes into new microcentrifuge tubes. Label these microcentrifuge tubes in the same way.
12. Add 500 μL methanol to the old tubes. Cap and vortex.
13. For a second time, transfer liquid from the bead tubes into the corresponding microcentrifuge tube. It is okay to transfer pulverized hair. This will eventually be centrifuged out.
14. Centrifuge the microcentrifuge tubes for 10 min at 2,800 x g.
15. Carefully remove the liquid and transfer it into new centrifuge tubes with corresponding labels. Be careful not to disturb or transfer the hair pellet.
16. Evaporate the liquid in the centrifuge tubes to dryness at 32 °C.
17. Reconstitute the samples by adding 200 μL of mobile phase A (HPLC-grade water with 1% formic acid) to the dry tubes. Vortex.
18. Transfer the liquid to amber vials with 250 μL inserts.

3. LC-MS/MS preparation

1. Make one liter of mobile phase A (HPLC-grade water with 1% formic acid) by adding some HPLC-grade water to a one-liter volumetric flask. Then add 10 mL of >95% formic acid to that flask, and then fill to the line with HPLC-grade water.
   1. Make one liter of mobile phase B (acetonitrile with 0.1% formic acid) by adding some acetonitrile to a one-liter volumetric flask. Then add 1 mL of >95% formic acid to that flask, and then fill to the line with acetonitrile.
2. Install a 2 x 100 mm column with 2.5 μm particle size and 100 Å pore size with polar endcapped, ether-linked phenyl beads fully made of porous silica in the column compartment. Ensure that column also has manufacturer recommended guard cartridge installed.
3. Open the data acquisition software and double-click Hardware Configuration. Highlight LCMS and click Activate Profile.
   1. Click New Sub-Project, or, if other sub-projects already exist, click Copy Sub-Project. Name the sub-project.
   2. Click New Document. Double-click Acquisition Method. Click Mass Spec within the Acquisition method window.
   3. Change the Scan Type dropdown to MRM (MRM). Make sure Polarity is set to Positive.
   4. Click Import List and select the .csv file MDR-TB LCMS method transitions.csv that is included in Supplemental Materials.
   5. Scroll down and set the Duration to 16.751 min. The appropriate cycle time and number of cycles will auto-populate.
   6. In left sidebar, click Integrated Valco Valve. Make sure that position name for step 0 is A. In Total Time (min) column, type in 0.4 in the first row and 13 in the second row.
   7. In the Position column, set row one to B and row two to A.
   8. In left sidebar, click Binary Pump. Set the gradient and flow rate table according to Table 4.
   9. In left sidebar, click Autosampler. Change injection volume to 10 μL. Click Temperature control enabled and set to 4 °C.
   10. In left sidebar, click Column Compartment. Set both right and left temperatures to 50 °C.
   11. Close and save method.
4. Create batch by clicking New Document and selecting Acquisition Batch. Type in a set name and select the newly created method from the dropdown bar.
   1. In a spreadsheet, create a batch that follows this order: calibration curve, quality controls, patient samples, calibration curve, quality controls, patient samples, calibration curve, quality controls. Add solvent blank injections at the start and end of the run, as well as before and after the calibration curve, quality controls and patient samples. Put at least eight solvent blank injections after injections of the calibration curve and high quality control vial in order to reduce analyte carryover.
      NOTE: More solvent blank injections may need to be added depending on column age.
   2. In the column adjacent to the sample names, type in the appropriate autosampler position for the corresponding vial.
   3. Click Add Set. In the pop-up window, type in the number of samples in the batch.
   4. Copy and paste sample names and vial locations from the spreadsheet to the newly created batch.
   5. Go to the Submit tab. Click Submit button.
5. Equilibrate system by inserting solvent line A into mobile phase A and solvent line B into mobile phase B. Open the purge valve on the binary pump.
   1. Set solvent composition to 50% B at 4 mL/min flow rate. Turn binary pump on.
   2. After 5 min, decrease flow to 0.3 mL/min. Close the purge valve. Check for any leaks.
   3. In the software, press Equilibrate on the top toolbar. Set time to >5 min, press OK.
   4. After the instrument has equilibrated, the modules in the bottom right of the window will appear green. Check that pressure has stabilized, and then start the batch by clicking Start Sample.

 4. Data analysis

1. After the batch is completed, open the quantitation software. Click the wand icon to create a new Results table.
   1. Click Browse to navigate to the appropriate folder, and then highlight the data file and click the right-pointing arrow to move the data into the Selected area. Click Next.
   2. Select Create New Method and click New. Input new quantitation method name and press Save and then Next.
   3. Select the first injection of the middle calibration point. Press Next.
   4. Tick mark all the transitions of the internal standards in the IS column.
   5. For the quantifier transitions for the reference standards, select the corresponding IS in the IS Name column. Click Next.
   6. Scroll through the transitions to assure that the automatically selected retention time is accurate. Make sure that Gaussian Smoothing is set to 1.5. All other default settings can remain as is (i.e., Noise Percentage 100%, Baseline Sub. Window 2.00 min, Peak Splitting 2 points).
      NOTE: If wanted, modify automatic integration parameters at this point. Since these parameters change based on instrument setup, we have not included ours here.
   7. Click Finish to apply the quantitation method to the batch.
2. Click the top left Displays the peak review button to view chromatograms. Navigate through the transitions using the left sidebar. Scroll through each injection of every quantifier transition and manually integrate the correct peak if necessary.
   1. To manually integrate a peak, click on the Enable manual integration mode button, zoom into the chromatogram by clicking and dragging along the x- or y-axis, and then draw a line from one baseline to the other baseline, defining the peak. Figure 3 shows two chromatograms: one that has INH, and therefore has been manually integrated, and another that does not have INH.
      NOTE: All injections must be integrated using the same parameters. Peak width can provide a guideline for adhering to these parameters, but sometimes peak width will differ. To quantify a peak, the retention time must be within ±0.15 min of the expected retention time for that analyte (as defined by the reference standard peaks), qualitatively confirmed as having the expected quantifier to qualifier ratio (as shown in Figure 2), and have a signal-to-noise ratio of greater than 10.
3. In the Sample Type column, set the calibration curve injections (with the exception of the blank calibration curve injections) to Standard. Set the quality control injections to Quality Control. Leave the remaining injections as Unknown.
   NOTE: This will be set across all transitions.
4. In the Actual Concentration column, type in the concentrations found in Table 5 for all calibration curve and quality control injections.
5. Click the second from top left Displays the calibration curve button. Click the Regression button.
6. Set Weighting Type to 1/x and press OK.
7. Validate the calibration curve and quality control samples to assure that the batch ran successfully.
   1. For each quantifier reference transition (not internal standard transitions), look at each calibration curve injection accuracy (in the Accuracy column). At least two-thirds of the calibration points must have an accuracy within 80-120%.
   2. For calibration points far outside of the expected accuracy, the injection may be an outlier. Exclude outliers if their calculated concentration is more than two standard deviations away from the other two injections of that vial. Clicking the “et peak to ‘not found button above each chromatogram.
   3. Check that the R-value displayed above the calibration curve is >0.975.
   4. Check that all quality control injections have an accuracy within 80-120%.
8. If all above conditions are satisfied, the batch has passed, and samples can be quantified. Click Edit in the toolbar, then click Copy entire table. Paste the table in a spreadsheet.
9. Take the average of the calculated concentration of the two sample injections to determine the reported concentration of each sample.

Results

An illustration of a chromatogram with confirmed levels of all 11 DR-TB drugs is shown in Figure 1. The retention time for each analyte can change when using different instruments and columns, so the exact retention time should be determined individually.

The Extracted Ion Chromatograms (EICs) for one particular drug (isoniazid, INH) in one of the calibrators (blank hair sample spiked with DR-TB drug reference standards) are shown in Figure 2<...

Discussion

We report here the protocol for the method we developed and validated for quantifying 11 anti-TB medications utilized in the treatment of DR-TB in small hair samples using LC-MS/MS. No other method for quantifying these 11 drugs in hair has been previously developed, validated and published. Our method can quantify sub-nanogram levels of drugs in only 20-30 hair strands of approximately 3 centimeters (cm) in length (~2 mg) and has already been validated²². The low weight of hair analyzed means tha...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2 mL injection vials	Agilent Technologies	5182-0716
250 uL injection vial inserts	Agilent Technologies	5181-8872
Bead ruptor 24	OMNI International	19001
Bead ruptor tubes (2 mL bead kit, 2.8mm ceramic, 2 mL microtubes)	OMNI International	19628
Bedaquiline	Toronto Research Chemicals	B119550
Bedaquiline-d6	Toronto Research Chemicals	B119552
Clofazimine	Toronto Research Chemicals	C324300
Clofazimine-d7	Toronto Research Chemicals	C324302
Disposable lime glass culture tubes	VWR	60825-425
Ethambutol	Toronto Research Chemicals	E889800
Ethambutol-d4	Toronto Research Chemicals	E889802
Ethionamide	Toronto Research Chemicals	E890420
Ethionamide-d5	ClearSynth	CS-O-06597
Formic acid	Sigma-Aldrich	F0507-100mL
Glass bottles	Corning	1395-1L
Hot Shaker	Bellco Glass Inc	7746-32110
HPLC	Agilent Technologies	Infinity 1260
HPLC grade acetonitrile	Honeywell	015-4
HPLC grade methanol	Honeywell	230-1L
HPLC grade water	Aqua Solutions Inc	W1089-4L
Isoniazid	Toronto Research Chemicals	I821450
Isoniazid-d4	Toronto Research Chemicals	I821452
LC column, Synergi 2.5 um Polar RP 100 A 100 x 2 mm	Phenomenex	00D-4371-B0
LC guard cartridge	Phenomenex	AJ0-8788
LC guard cartridge holder	Phenomenex	AJ0-9000
LC-MS/MS quantitation software	Sciex	Multiquant 2.1
Levofloxacin	Sigma-Aldrich	1362103-200MG
Levofloxacin-d8	Toronto Research Chemicals	L360002
Linezolid	Toronto Research Chemicals	L466500
Linezolid-d3	Toronto Research Chemicals	L466502
Micro centrifuge tubes	E&K Scientific	695554
Moxifloxacin	Toronto Research Chemicals	M745000
Moxifloxacin-13C, d3	Toronto Research Chemicals	M745003
MS/MS	Sciex	Triple Quad 5500
OPC 14714	Toronto Research Chemicals	O667600
Pretomanid (PA-824)	Toronto Research Chemicals	P122500
Prothionamide	Toronto Research Chemicals	P839100
Prothionamide-d5	Toronto Research Chemicals	P839102
Pyrazinamide	Toronto Research Chemicals	P840600
Pyrazinamide-15N, d3	Toronto Research Chemicals	P840602
Septum caps for injection vials	Agilent Technologies	5185-5862
Turbovap LV evaporator	Biotage	103198/11