The authors would like to thank Professor Keertan Dheda, Dr. Ali Esmail, and Marietjie Pretorius at the University of Cape Town Lung Institute who facilitated the collection of hair samples for the study. The authors further gratefully acknowledge the contributions of the participants of this study.

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
<ol>
	<li>Obtain written informed consent.</li>
	<li>Use clean scissors to cut approximately 20-30 scalp hair strands from the occipital region as close to the scalp as possible.</li>
	<li>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.</li>
	<li>In addition to patient samples, collect &#8220;blank hair&#8221;: a scalp hair sample from someone who has not taken TB medication. Collect a large amount (&#62;30 mg blank hair for each 20 patient samples).</li>
</ol>
2. Drug extraction
<ol>
	<li>Label bead tubes. Each patient sample requires one tube. Label 12 tubes from &#8220;C0&#8221; to &#8220;C11&#8221;, 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.</li>
	<li>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.</li>
	<li>Weigh 2 mg of the hair sample into a bead tube.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Make internal standard mix and add it to the samples.
	<ol>
		<li>Add ~40 mL of methanol to a 50 mL volumetric flask.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Cap and mix the volumetric flask. Add 500 &#956;L of the mixture to each of the pulverized hair tubes, except for the matrix blank tube. Add 500 &#956;L methanol to the matrix blank tube.</li>
	</ol>
	</li>
	<li>Make reference standard mixes.
	<ol>
		<li>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.</li>
		<li>Add 1768 &#956;L 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 &#8220;Ref Std Mix 1x&#8221;. Vortex.</li>
		<li>Spike 100 &#956;L from &#8220;Ref Std Mix 1x&#8221; into 900 &#956;L methanol in a new vial. Label this vial &#8220;Ref Std Mix 10x&#8221;. Vortex.</li>
		<li>Spike 100 &#956;L from &#8220;Ref Std Mix 10x&#8221; into 900 &#956;L methanol in a new vial. Label this vial &#8220;Ref Std Mix 100x&#8221;. Vortex.</li>
		<li>Spike 100 &#956;L from &#8220;Ref Std Mix 100x&#8221; into 900 &#956;L methanol in a new vial. Label this vial &#8220;Ref Std Mix 1000x&#8221;. Vortex.</li>
	</ol>
	</li>
	<li>Spike the calibration curve tubes by adding the amount of Ref Std Mix described in Table 3.</li>
	<li>Create QC mixes and spike quality control tubes.
	<ol>
		<li>Label 5 vials &#8220;QC-A&#8221; through &#8220;QC-E&#8221;.</li>
		<li>Add the following amounts of methanol to the five labeled vials: 
		QC-A: 990 &#956;L 
		QC-B: 940 &#956;L 
		QC-C: 950 &#956;L 
		QC-D: 980 &#956;L 
		QC-E: 950 &#956;L 
		&#160;</li>
		<li>Using the 1 mg/mL drug stocks created in step 2.7.1, add 10 &#956;L to the specific vials listed below. For the BDQ and CLF stocks, which are at 0.5 mg/mL, add 20 &#956;L 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.</li>
		<li>Label a vial as &#8220;QC-A df100&#8221;. Dilute 10 &#956;L of QC-A into 990 &#956;L methanol.</li>
		<li>Label a vial as &#8220;Low QC stock&#8221;. Add 1832 &#956;L methanol to this vial. Add the amounts of QC mixes detailed below: 
		QC-A df100: 80 &#956;L 
		QC-B: 8 &#956;L 
		QC-C: 80 &#956;L 
		&#160;</li>
		<li>Label a vial as &#8220;Mid QC stock&#8221;. Add 760 &#956;L methanol to this vial. Add the amounts of QC mixes detailed below: 
		QC-A df100: 800 &#956;L 
		QC-B: 40 &#956;L 
		QC-C: 400 &#956;L 
		&#160;</li>
		<li>Label a vial as &#8220;High QC stock&#8221;. Add 1376 &#956;L methanol to this vial. Add the amounts of QC mixes detailed below: 
		QC-D: 160 &#956;L 
		QC-E: 320 &#956;L 
		MFX, 1mg/mL stock: 16 &#956;L 
		INH, 1mg/mL stock: 32 &#956;L 
		LFX, 1mg/mL stock: 32 &#956;L 
		LZD, 1mg/mL stock: 32 &#956;L 
		PZA, 1mg/mL stock: 32 &#956;L 
		&#160;</li>
		<li>Spike 10 &#956;L Low QC stock into the Low QC bead tube.</li>
		<li>Spike 10 &#956;L Mid QC stock into the Mid QC bead tube.</li>
		<li>Spike 10 &#956;L High QC stock into the High QC bead tube.</li>
	</ol>
	</li>
	<li>Place all tubes in hot shaker for 2 h at 37 &#176;C. Shaking should be slow enough that the water does not splash up on to the tubes.</li>
	<li>Remove tubes from shaker. Transfer liquid from bead tubes into new microcentrifuge tubes. Label these microcentrifuge tubes in the same way.</li>
	<li>Add 500 &#956;L methanol to the old tubes. Cap and vortex.</li>
	<li>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.</li>
	<li>Centrifuge the microcentrifuge tubes for 10 min at 2,800 x g.</li>
	<li>Carefully remove the liquid and transfer it into new centrifuge tubes with corresponding labels. Be careful not to disturb or transfer the hair pellet.</li>
	<li>Evaporate the liquid in the centrifuge tubes to dryness at 32 &#176;C.</li>
	<li>Reconstitute the samples by adding 200 &#956;L of mobile phase A (HPLC-grade water with 1% formic acid) to the dry tubes. Vortex.</li>
	<li>Transfer the liquid to amber vials with 250 &#956;L inserts.</li>
</ol>
3. LC-MS/MS preparation
<ol>
	<li>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 &#62;95% formic acid to that flask, and then fill to the line with HPLC-grade water.
	<ol>
		<li>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 &#62;95% formic acid to that flask, and then fill to the line with acetonitrile.</li>
	</ol>
	</li>
	<li>Install a 2 x 100 mm column with 2.5 &#956;m particle size and 100 &#197; 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.</li>
	<li>Open the data acquisition software and double-click Hardware Configuration. Highlight LCMS and click Activate Profile.
	<ol>
		<li>Click New Sub-Project, or, if other sub-projects already exist, click Copy Sub-Project. Name the sub-project.</li>
		<li>Click New Document. Double-click Acquisition Method. Click Mass Spec within the Acquisition method window.</li>
		<li>Change the Scan Type dropdown to MRM (MRM). Make sure Polarity is set to Positive.</li>
		<li>Click Import List and select the .csv file MDR-TB LCMS method transitions.csv that is included in Supplemental Materials.</li>
		<li>Scroll down and set the Duration to 16.751 min. The appropriate cycle time and number of cycles will auto-populate.</li>
		<li>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.</li>
		<li>In the Position column, set row one to B and row two to A.</li>
		<li>In left sidebar, click Binary Pump. Set the gradient and flow rate table according to Table 4.</li>
		<li>In left sidebar, click Autosampler. Change injection volume to 10 &#956;L. Click Temperature control enabled and set to 4 &#176;C.</li>
		<li>In left sidebar, click Column Compartment. Set both right and left temperatures to 50 &#176;C.</li>
		<li>Close and save method.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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.</li>
		<li>In the column adjacent to the sample names, type in the appropriate autosampler position for the corresponding vial.</li>
		<li>Click Add Set. In the pop-up window, type in the number of samples in the batch.</li>
		<li>Copy and paste sample names and vial locations from the spreadsheet to the newly created batch.</li>
		<li>Go to the Submit tab. Click Submit button.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Set solvent composition to 50% B at 4 mL/min flow rate. Turn binary pump on.</li>
		<li>After 5 min, decrease flow to 0.3 mL/min. Close the purge valve. Check for any leaks.</li>
		<li>In the software, press Equilibrate on the top toolbar. Set time to &#62;5 min, press OK.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
&#160;4. Data analysis
<ol>
	<li>After the batch is completed, open the quantitation software. Click the wand icon to create a new Results table.
	<ol>
		<li>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.</li>
		<li>Select Create New Method and click New. Input new quantitation method name and press Save and then Next.</li>
		<li>Select the first injection of the middle calibration point. Press Next.</li>
		<li>Tick mark all the transitions of the internal standards in the IS column.</li>
		<li>For the quantifier transitions for the reference standards, select the corresponding IS in the IS Name column. Click Next.</li>
		<li>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.</li>
		<li>Click Finish to apply the quantitation method to the batch.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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 &#177;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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>In the Actual Concentration column, type in the concentrations found in Table 5 for all calibration curve and quality control injections.</li>
	<li>Click the second from top left Displays the calibration curve button. Click the Regression button.</li>
	<li>Set Weighting Type to 1/x and press OK.</li>
	<li>Validate the calibration curve and quality control samples to assure that the batch ran successfully.
	<ol>
		<li>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%.</li>
		<li>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 &#8220;et peak to &#8216;not found button above each chromatogram.</li>
		<li>Check that the R-value displayed above the calibration curve is &#62;0.975.</li>
		<li>Check that all quality control injections have an accuracy within 80-120%.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>Take the average of the calculated concentration of the two sample injections to determine the reported concentration of each sample.</li>
</ol>

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This work was supported by the National Institute of Allergy and Infectious Diseases RO1 AI123024 (Co-PIs: John Metcalfe and Monica Gandhi).

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...

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 globally1,2. 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 providers5,6. Pill counts and medication event monitoring systems (MEMS) can be more objective7 but do not measure actual drug consumption8,9,10. Drug levels in biomatrices can provide both adherence and pharmacokinetic data. Therefore, plasma drug levels are commonly used in therapeutic drug monitoring11,12. 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. &#8220;White coat&#8221; 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 exposure14,15. 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 infection17,18,19,20,21. 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&#8217;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.

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

validated lc-ms/ms panel for quantifying 11 drug-resistant tb medications in small hair samples

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&#8217;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.

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<...

Watch this Scientific Journal Video about Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples at JoVE.com

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

Current methods of analyzing patients&#8217; 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.

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...

validated-lc-msms-panel-for-quantifying-11-drug-resistant-tb

Research

JoVE Journal

Medicine

7.7K Views.  University of California San Francisco. Therapietrouw is zeer uitdagend en medicijnmetabolisme kan variëren van persoon tot persoon, zodat objectieve meting van de drug niveaus kan blijken te zijn belangrijk bij het voorkomen van het ontstaan van resistentie tijdens tbc behandeling. Als therapietrouw nauwkeuriger en gemakkelijker kan worden gekwantificeerd, dan kunnen zorgverleners een behandelplan ontwikkelen dat de ontwikkeling van resistentie tegen geneesmiddelen zou beperken. We hebben het eerste meervoudige analyt massaspectr...

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

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Biochemistry

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Immunology and Infection

The MODS method for diagnosis of tuberculosis and multidrug resistant tuberculosis

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and preventing new infections.  Furthermore, the emergence of multidrug resistant tuberculosis (MDRTB) means that detection of drug resistance is necessary for stopping the spread of drug-resistant strains.  The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of TB and MDRTB.  The MODS assay is based on three principles: 1) mycobacterium tuberculosis (MTB) grows faster in liquid media than on solid media 2) microscopic MTB growth can be detected earlier in liquid media than waiting for the macroscopic appearance of colonies on solid media, and that growth is characteristic of MTB, allowing it to be distinguished from atypical mycobacteria or fungal or bacterial contamination 3) the drugs isoniazid and rifampicin can be incorporated into the MODS assay to allow for simultaneous direct detection of MDRTB, obviating the need for subculture to perform an indirect drug susceptibility test. Competing current diagnostics are hampered by low sensitivity with sputum smear, long delays until diagnosis with solid media culture, prohibitively high cost with existing liquid media culture methods, and the need to do subculture for indirect drug susceptibility testing to detect MDRTB.  In contrast, the non-proprietary MODS method has a high sensitivity for TB and MDRTB, is a relatively rapid culture method, provides simultaneous drug susceptibility testing for MDRTB, and is accessible to resource-limited settings at just under $3 for testing for TB and MDRTB.

The microscopic-observation drug-susceptibility (MODS) assay is a low-cost, low-tech tool for high-performance detection of tuberculosis (TB) and multidrug-resistant tuberculosis (MDRTB). This video describes the MODS liquid media culture method.

Patients with active pulmonary tuberculosis (TB) infect 10-15 other persons per year, making diagnosing active TB essential to both curing the patient and ...

Biology

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

Chemistry

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United States. Tacrolimus is a narrow therapeutic index drug and as such requires therapeutic drug monitoring and dose adjustment based on its whole blood trough concentrations. To facilitate home therapeutic drug and adherence monitoring, the collection of dried blood spots is an attractive concept. After a finger stick, the patient collects a blood drop on filter paper at home. After the blood is dried, it is mailed to the analytical laboratory where tacrolimus is quantified using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) in combination with a simple manual protein precipitation step and online column extraction.
For tacrolimus analysis, a 6-mm disc is punched from the saturated center of the blood spot. The blood spot is homogenized using a bullet blender and then proteins are precipitated with methanol/0.2 M ZnSO4 containing the internal standard D2,13C-tacrolimus. After vortexing and centrifugation, 100 &#181;l of supernatant is injected into an online extraction column and washed with 5 ml/min of 0.1 formic acid/acetonitrile (7:3, v:v) for 1 min. Hereafter, the switching valve is activated and the analytes are back-flushed onto the analytical column (and separated using a 0.1% formic acid/acetonitrile gradient). Tacrolimus is quantified in the positive multi reaction mode (MRM) using a tandem mass spectrometer.
The assay is linear from 1 to 50 ng/ml. Inter-assay variability (3.6%-6.1%) and accuracy (91.7%-101.6%) as assessed over 20 days meet acceptance criteria. Average extraction recovery is 95.5%. There are no relevant carry-over, matrix interferences and matrix effects. Tacrolimus is stable in dried blood spots at RT and at +4 &#176;C for 1 week. Extracted samples in the autosampler are stable at +4 &#176;C for at least 72 hr.

Here we describe a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay to quantify the immunosuppressant tacrolimus in dried blood spots using a simple manual protein precipitation step and online column extraction.

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

Harvesting the Orthogonality of LC and TIMS&#45;TOF MS&#47;MS to Screen Fentanyl Analogs

Fentanyl Analog Screening using LC-TIMS-TOF MS/MS

The use of fentanyl and the emergence of fentanyl analogs over recent decades has become an increasing concern to the community at large. Fentanyl and its analogs are the major contributors to fatal and nonfatal overdoses in the United States. Most recent cases of fentanyl-related overdose are linked to illicitly manufactured fentanyl and its associated extreme potency. In the present work, we describe a high-throughput analytical protocol for the screening of fentanyl analogs. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and tandem mass spectrometry allow for the separation and assignment of hundreds of fentanyl analogs from a single sample in a single scan. The described approach takes advantage of the recent development of data-dependent acquisition and data-independent acquisition using parallel accumulation in the mobility trap followed by sequential fragmentation using collision-induced dissociation. The fentanyl analogs are confidently assigned based on their retention time, mobility, and MS fragmentation pattern.

Author Spotlight: An Efficient Methodology to Confidently Differentiate and Characterize Fentanyl Analogs 

A method for the screening of fentanyl analogs based on their retention time, mobility, and mass spectrometry fragmentation pattern.

The use of fentanyl and the emergence of fentanyl analogs over recent decades has become an increasing concern to the community at large. Fentanyl and its ...

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers&#8217; safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 &#176;C, 85 &#176;C, or 95 &#176;C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 &#181;L of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of &#60;1 log10eCFU/mL (i.e., &#60;10 CFU/mL). Further studies will determine the proportion of very low burden patients that cause false negative results due to heat inactivation.

A Tuberculosis Molecular Bacterial Load Assay TB-MBLA

We describe a tuberculosis molecular bacterial load assay test performed after heat inactivation of sputum. Heat inactivation renders sputum samples noninfectious and obviates the need for containment level 3 laboratories for tuberculosis molecular tests.

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

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