The authors thank Dr. Sch&#252;tze for his help with establishing the presented method and Dr. Zoller for the valuable input regarding the proper calibration range. The authors also acknowledge the technical staff of the mass spectrometry facility.

NOTE: It is recommended to work in a fume hood when handling organic solvent, such as methanol. Prepare all buffers and mobile phases in volumetric flasks. If not otherwise specified, the solutions can be stored at room temperature for up to 1 month after preparation.
1. Preparation of the Calibrators and Quality Control Samples
NOTE: A corresponding data analysis sheet for the preparation of stock and spike solutions is given in the Supplemental File. For reasons of traceability, insert the manufacturer, catalog number, and a lot number of each antibiotic in the corresponding columns. Dissolve all antibiotics in a cold storage at 4 &#176;C and keep the working time as short as possible.
<ol>
	<li>Prepare 100 mL of 25% methanol in water: prefill a 100 mL volumetric flask with 25 mL of absolute methanol and fill it up to 100 mL with distilled water.</li>
	<li>Prepare 10 mL of 200 mM acetic acid in water: prefill a 10 mL volumetric flask with 9 mL of HPLC grade water, add 115 &#181;L of glacial acetic acid (99.5% purity, 17.4 M), and add distilled water up to 10 mL.</li>
	<li>Prepare 25 mL of 25% methanol in water with 20 mM acetic acid: prefill a 25 mL volumetric flask with 2.5 mL of the aqueous 200 mM acetic acid solution, add 6.25 mL of absolute methanol, and fill up the flask to 25 mL with distilled water.</li>
	<li>Use a precision scale to weigh the proper amounts of antibiotics in 15-mL conical tubes as described in the Supplemental File in the column initial weight.</li>
	<li>Prepare stock solutions of the fluoroquinolones, ciprofloxacin, and moxifloxacin in the 25% methanol-water including 20 mM acetic acid. To do this, add the corresponding volume to the weighted quantities as described in the Supplemental File in the column &#34;final volume&#34;. Rapidly dissolve the fluoroquinolone antibiotics in an ultrasound bath for 2 min and by intense vortexing.</li>
	<li>Prepare stock solutions of cefepime, meropenem, linezolid, and piperacillin in the 25% methanol-water. To do this, add the corresponding volume to the weighted quantities as described in the Supplemental File in the column final volume and rapidly dissolve the antibiotics by intense vortexing. Dissolve meropenem as the last substance.</li>
	<li>Combine the stock solutions of all the antibiotics as described in the corresponding volume of stock solution chart in the Supplemental File to yield tenfold concentrated spike-solutions.</li>
	<li>Spike nine volumes of drug-free serum with one volume of the tenfold concentrated spike solutions to obtain the serum calibrators 0&#8211;7 and quality controls (QC) A&#8211;D. For example, add 0.5 mL of spike solution to 4.5 mL of serum in a 10-mL polypropylene tube and incubate it for 15 min in the cold storage at 4 &#176;C on a roller mixer at 50 rpm.</li>
	<li>Use a repetitive pipette to generate 100 &#181;L aliquots of the calibrators and QCs in 1.5 mL polypropylene tubes.</li>
	<li>Store the calibrators, quality controls, and antibiotic stock solutions at -80 &#176;C for up to six months.</li>
	<li>For each antibiotic, also prepare a neat solution containing 1,000 mg/L of a single antibiotic. Dilute the corresponding stock solution with an appropriate diluent (e.g., for ciprofloxacin, use 25% methanol-water including 20 mM acetic acid). 
	NOTE: The neat antibiotic solutions are required for the instrument-tuning only.</li>
</ol>
2. Preparation of the Internal Standards Mix
NOTE: Internal standards are isotope-labeled counterparts of the analytes of interest that are added to a sample during sample cleanup. As the internal standards have almost identical overall physicochemical properties to their unlabeled counterparts, they compensate for the matrix effects of a given sample.
<ol>
	<li>Prepare 10 mL of 50% methanol in water by adding 5 mL of absolute methanol to a 10 mL shake flask and fill it up to 10 mL with distilled water.</li>
	<li>Prepare 10 mL of 50% methanol in water including 20 mM acetic acid. To do this, add 1 mL of 200 mM acetic acid to a 10 mL flask, add 5 mL of absolute methanol, and fill it up to 10 mL with distilled water.</li>
	<li>Generate stock solutions of internal standards (IS) with 1,000 mg/L directly in the vials provided by the manufacturer. Dissolve cefepime-13C12D3 sulfate in distilled water, meropenem-D6, linezolid-D3, and piperacillin-D5 in a 50% methanol-water solution. Dissolve ciprofloxacin-D8 in 50% methanol-water with 20 mM acetate and moxifloxacin hydrochloride-13C1D3 in distilled water with 20 mM acetate.</li>
	<li>Combine the IS stock solutions in a 1.5 mL polypropylene tube to yield a fivefold concentrated internal standard mix. Add 10 &#181;L of cefepime-13C12D3, 10 &#181;L of meropenem-D6, 1 &#181;L of ciprofloxacin-D8, 2 &#181;L of moxifloxacin hydrochloride-13C1D3, 2 &#181;L of linezolid-D3, and 10 &#181;L of piperacillin-D5 to 965 &#181;L of 25% methanol-water.</li>
	<li>Store the internal standard stock solutions and the fivefold concentrated IS mix at -80 &#176;C.</li>
</ol>
3. Patient Sample Storage
NOTE: Ensure that the serum is obtained as fast as possible and that the cold chain of frozen samples is maintained.
<ol>
	<li>Collect the whole blood in the serum collection tubes.</li>
	<li>Let the blood clot for 20&#8211;30 min at room temperature.</li>
	<li>Separate the serum from the blood by centrifugation at 2,000 x g for 10 min.</li>
	<li>Transfer the supernatant to a clean polypropylene tube.</li>
	<li>Store the serum up to six months at -80 &#176;C until it is assayed. Alternatively, store the samples up to 3 days at -20 &#176;C.</li>
</ol>
4. Buffer Preparation for Chromatography
<ol>
	<li>To prepare 1 M ammonium formate in water, dissolve 6.306 g of ammonium formate in 100 mL of HPLC grade water using a 100 mL shake flask. Store the solution up to 1 month at 4 &#176;C.</li>
	<li>Prepare the mobile phase A [10 mM ammonium formate in water-formic acid (99.9:0.1 v/v)]. Prefill a 1,000 mL volumetric flask with approximately 500 mL of HPLC grade water, add 1 mL of formic acid and 10 mL of the 1 M ammonium formate solution, and fill it to 1,000 mL with HPLC grade water. Transfer mobile phase A to a clean glass bottle and connect it to the HPLC system. Store mobile phase A up to 2 weeks at room temperature.</li>
	<li>Prepare the mobile phase B. Transfer HPLC-grade absolute methanol into a clean glass bottle and connect it to the HPLC system.</li>
	<li>Use absolute methanol as the needle wash solvent and connect the corresponding tube to the glass bottle containing mobile phase B.</li>
	<li>Generate the seal and a purge solvent of methanol-water-formic acid (7:92.9:0.1, v/v/v). Prefill a 1,000 mL volumetric flask with approximately 500 mL of distilled water, add 70 mL of absolute methanol, 1 mL of formic acid, and add distilled water to 1,000 mL. Transfer the solvent to a clean glass bottle and connect it with the HPLC system. 
	Note: Various autosampler systems use both a strong and a weak needle wash solvent. In such a case, prepare the wash solutions according to the manufacturer&#8217;s recommendations. For example, do the strong wash with methanol-water-isopropylic alcohol (70:20:10, v/v/v) and the weak wash with water-methanol (95:5, v/v).</li>
</ol>
5. Instrument Tuning
NOTE: This step is performed for the set-up of the method on a specific mass spectrometer.
<ol>
	<li>Dilute the neat 1,000 mg/L analyte and the internal standard solutions 1:10 or 1:100 in a mixture of mobile phase A and B (50:50, v/v), depending on the detector signal intensities. Tune the mass spectrometer with the autotune function or do a manual tuning for the following parent-to-daughter ions transitions14: cefepime (481.0 &#62; 167.0/395.7), cefepime-13C12D3 (485.1 &#62; 167.1/400.0), meropenem (384.1 &#62; 114.0/141.0), meropenem-D6 (390.1 &#62; 114.0/147.2), ciprofloxacin (332.0 &#62; 231.0/245.0), ciprofloxacin-D8 (340.1 &#62; 235.1/249.3), moxifloxacin (402.0 &#62; 261.0/383.9), moxifloxacin-13C1D3 (406.1 &#62; 265.1/388.0), linezolid (338.0 &#62; 235.0/296.0), linezolid-D3 (341.1 &#62; 235.1/297.1), piperacillin (518.0 &#62; 143.0/358.9), and piperacillin-D5 (523.1 &#62; 142.8/364.1).</li>
	<li>For instruments with autotuning, use the autotune function to automatically adjust the voltage and settings of the MS inlet through the detectors.</li>
	<li>For instruments with manual tuning, adjust the settings (e.g., collision voltage and collision energy) until the optimum (usually the maximum) signal intensity is obtained at the detector for each parent and daughter ion. For example, plug a mixing tee, deliver mobile phase A and B (50:50, v/v) at 0.5 mL/min, and continuously infuse the neat antibiotic or internal standard with a flow rate of 0.1 mL/min.</li>
</ol>
6. HPLC-MS/MS Set-up
NOTE: Features of the mass spectrometer, HPLC system (including the autosampler), and the corresponding software depend on the manufacturer. Adapt the mass spectrometer parameters and the wash procedure according to the manufacturer&#8217;s recommendations.
<ol>
	<li>Store the mass spectrometer parameters in a corresponding &#39;MS tune file&#39;. Use electrospray ionization in the positive mode (ESI+) for all analytes. Adapt the ion source settings for the instrument used (e.g., a capillary voltage of 1.5 kV, a source temperature of 120 &#176;C, a desolvation temperature of 400 &#176;C, a desolvation gas flow rate of 600 L/h, an RF lens voltage of 0.1 V, and a dwell time of 80 ms).</li>
	<li>Specify the analyte and internal standards tune parameters (e.g., capillary voltage, collision energy) in an &#39;MS file&#39;.</li>
	<li>Set the autosampler conditions as follows in the &#39;inlet file&#39;: the sample temperature at 10 &#176;C with a limit of &#177; 5 &#176;C; the wash sequence at 1x purge-wash-purge with a 600 &#181;L purge volume replacement.</li>
	<li>In the above-mentioned &#39;inlet file&#39;, set the flow rate to 0.4 or 0.5 mL/min, the run time to 4 min, the pressure high limit to 345 bar, and the column temperature to 30 &#176;C with a limit of &#177; 5 &#176;C. Add the solvent name of mobile phases A and B and set them to 7% B/93% A, respectively.</li>
	<li>Program the chromatographic gradient in the &#39;inlet file&#39; as follows: 0.00&#8211;0.10 min with 7% mobile phase B/93% A, 0.11&#8211;0.60 min with 65% mobile phase B/35% A, 0.61&#8211;2.10 min with 95% mobile phase B/5% A, 2.11&#8211;4.00 min with 7% mobile phase B/93% A. 
	NOTE: Calculate the extra-column volume, the hold-up volume for the instrumental platform, and the analyte retention factors as described in the USP &#60;621&#62; Chromatography guideline16.</li>
</ol>
7. Sample Measurement Master File
NOTE: With the &#39;sample measurement master file&#39;, the patient samples are specified, the HPLC-MS/MS analysis is started, and the data evaluation is performed. Two separate template files including a low- and high-quality control pair are generated; one template includes QC pair A and C, the other one QC pair B and D.
<ol>
	<li>Create a new &#39;sample measurement master file&#39;. Select the above-mentioned &#39;MS tune file&#39;, &#39;MS file&#39;, and&#160;&#39;inlet file&#39;&#160;(section 6), insert them in each sample line, and specify the injection volume with 15 &#181;L.</li>
	<li>In ascending order, add the &#8220;sample text&#8221; for calibrators 0&#8211;7 and quality control (QC) pair A/C or QC pair B/D.</li>
	<li>Specify the sample type. Select the sample type &#8220;standard&#8221; for the calibrators and &#8220;QC&#8221; for the quality control pairs.</li>
	<li>Specify the concentration of each antibiotic substance for the corresponding calibrators and quality controls (see the spreadsheet, concentration [&#181;g/mL] Cal 7&#8211;Cal 0, QC A/C or B/D,).</li>
	<li>Program the &#39;data evaluation method&#39;.&#160;Use the transitions that were optimized during the instrument tuning (section 5). Match each antibiotic with the corresponding isotope-labeled standard (e.g., meropenem - meropenem-D6).</li>
</ol>
8. Sample Cleanup and HPLC-MS/MS Analysis
NOTE: For each sample batch, a paired quality control set with a low and high antibiotic concentration (QC A/C or QC B/D) is processed and analyzed. Between different batches, the paired QC samples are used in an alternate sequence (e.g., on day 1, select the &#39;sample measurement master file&#39; including QC pair A/C; on day 2, select the one including QC pair B/D. The processing of the serum samples is illustrated in Figure 1.
<ol>
	<li>Prepare the precipitation agent 10% methyl-tert-butyl ether in methanol (10:90, v/v) (e.g., prefill a 25-mL volumetric flask with 2.5 mL of methyl-tert-butyl ether and fill it to 25 mL with absolute methanol).</li>
	<li>Place the C8 reverse phase into the column chamber. Connect it to the HPLC and mass spectrometer in the direction of the flow.</li>
	<li>Generate the sample list. Open the corresponding &#39;sample measurement master file&#39; template and add the patient samples meant to be processed to the list. Generate groups of up to 20 patient samples and flank them with the corresponding quality control pair.</li>
	<li>Wet-prime the HPCL system using the &#39;inlet file&#39; control software: set the &#8220;wet prime&#8221; function to 50% mobile phase A/50% B, and wet-prime for 2 min with a flow rate of 1 mL/min.</li>
	<li>Refresh the syringe. To do this, execute 6 strokes of 600 &#181;L in the control software.</li>
	<li>Equilibrate the C8 reverse phase column. Using the software, turn on the flow in the &#39;inlet file&#39; and flush it with 7% mobile phase B/93% A for a minimum of 5 min, using a flow rate of 0.5 mL/min. Verify the column temperature of 30 &#176;C.</li>
	<li>Thaw the patient samples, one aliquot of calibrators 0&#8211;7, and a quality control pair (either A/B or C/D).</li>
	<li>With a repetitive pipette, add 25 &#181;L of the internal standard mix to the 100 &#181;L calibrator, QC sample, or patient serum in a 1.5 mL polypropylene tube, and vortex the tube for a few seconds.</li>
	<li>Incubate the mixture for 5 min at room temperature on a benchtop shaker (e.g., at 1,200 rpm).</li>
	<li>With a repetitive pipette, add 150 &#181;L of a precipitation reagent to the sample-internal standard mix.</li>
	<li>Again, vortex the tube for a few seconds and incubate it for 5 min at room temperature on a benchtop shaker (e.g., at 1,200 rpm).</li>
	<li>Centrifuge the suspension at 20,000 x g in a tabletop centrifuge for 10 min at 4 &#176;C.</li>
	<li>Dilute the supernatant 1:3 with HPLC grade water using a glass vial with a micro-insert and load it as processed samples to the autosampler.</li>
	<li>Manually start the HPLC-MS/MS analysis in the &#39;sample measurement control file&#39;. 
	NOTE: For prolonged storage, thoroughly flush the analytical column according to the manufacturer&#8217;s recommendation [e.g., 0.5 mL/min methanol-water (50:50, v/v)] to prevent phase collapse.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58148/58148fig1.jpg" /> 
Figure 1: Schematic representation of the sample cleanup. Protein precipitation at the high centrifugal force gives a dense pellet and clear supernatant, indicating that protein precipitation was complete. The entire processing time is approximately 30 min, including the sample cleanup, the chromatographic separation, and the MS/MS analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58148/58148fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
9. Quality Assessment and Quantification
<ol>
	<li>To process the samples, open the corresponding &#39;sample measurement control file&#39;, select the calibrators, quality controls, and patient samples, and evaluate them with the &#39;antibiotics quantification method&#39;. </li>
	<li>Check whether the peaks for a specific analyte are properly integrated. Inspect the peaks for each calibrator, QC, and patient sample, and manually reintegrate them at the baseline if necessary.</li>
	<li>Study the calibration curve and examine whether it fulfills the following quality criteria: a) linearity over the entire calibration range, b) a calibration coefficient r2 &#62; 0.995, c) the deviation of each calibration standard within &#177; 15% of the nominal value, except for the lower limit of quantification (LLOQ), where &#177; 20% is required.</li>
	<li>Reject a calibration standard not complying with the above-mentioned criteria and re-evaluate the calibration curve, including the regression analysis.</li>
	<li>Study the quality controls and examine whether the deviations are within &#177; 15% of the nominal value.</li>
	<li>If the concentration of a patient sample exceeds the concentration of the highest calibrator, dilute the sample with distilled water, up to 1:5 (e.g., 100 &#181;L of serum plus 400 &#181;L of distilled water) before the sample cleanup. Reperform steps 8.8&#8211;8.14 for that specific sample and reprocess it.</li>
</ol>

<ol>
	<li>Fleischmann, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+Global+Incidence+and+Mortality+of+Hospital-treated+Sepsis.+Current+Estimates+and+Limitations.">Assessment of Global Incidence and Mortality of Hospital-treated Sepsis. Current Estimates and Limitations.</a> American Journal of Respiratory and Critical Care Medicine. 193 (3), 259-272 (2016).</li><li>Dellinger, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surviving+Sepsis+Campaign:+international+guidelines+for+management+of+severe+sepsis+and+septic+shock,+2012.">Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock, 2012.</a> Intensive Care Medicine. 39 (2), 165-228 (2013).</li><li>Lodise, T. P., Drusano, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+and+pharmacodynamics:+optimal+antimicrobial+therapy+in+the+intensive+care+unit.">Pharmacokinetics and pharmacodynamics: optimal antimicrobial therapy in the intensive care unit.</a> Critical Care Clinics. 27 (1), 1-18 (2011).</li><li>Macedo, R. S., Onita, J. H., Wille, M. P., Furtado, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacokinetics+and+pharmacodynamics+of+antimicrobial+drugs+in+intensive+care+unit+patients.">Pharmacokinetics and pharmacodynamics of antimicrobial drugs in intensive care unit patients.</a> Shock. 39, 24-28 (2013).</li><li>Petersson, J., Giske, C. G., Eliasson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+dosing+of+piperacillin+and+meropenem+fail+to+achieve+adequate+plasma+concentrations+in+ICU+patients.">Standard dosing of piperacillin and meropenem fail to achieve adequate plasma concentrations in ICU patients.</a> Acta Anaesthesiologica Scandinavica. 60 (10), 1425-1436 (2016).</li><li>Abdul-Aziz, M. H., Lipman, J., Mouton, J. W., Hope, W. W., Roberts, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+pharmacokinetic/pharmacodynamic+principles+in+critically+ill+patients:+optimizing+efficacy+and+reducing+resistance+development.">Applying pharmacokinetic/pharmacodynamic principles in critically ill patients: optimizing efficacy and reducing resistance development.</a> Seminars in Respiratory and Critical Care Medicine. 36 (1), 136-153 (2015).</li><li>Imani, S., Buscher, H., Marriott, D., Gentili, S., Sandaradura, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Too+much+of+a+good+thing:+a+retrospective+study+of+beta-lactam+concentration-toxicity+relationships.">Too much of a good thing: a retrospective study of beta-lactam concentration-toxicity relationships.</a> Journal of Antimicrobial Chemotherapy. 72 (10), 2891-2897 (2017).</li><li>Ventola, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibiotic+resistance+crisis:+part+1:+causes+and+threats.">The antibiotic resistance crisis: part 1: causes and threats.</a> Pharmacy &amp; Therapeutics. 40 (4), 277-283 (2015).</li><li>Pulcini, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+stewardship:+update+and+perspectives.">Antibiotic stewardship: update and perspectives.</a> Clinical Microbiology and Infection. 23 (11), 791-792 (2017).</li><li>Cairns, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+a+multidisciplinary+antimicrobial+stewardship+team+on+the+timeliness+of+antimicrobial+therapy+in+patients+with+positive+blood+cultures:+a+randomized+controlled+trial.">The impact of a multidisciplinary antimicrobial stewardship team on the timeliness of antimicrobial therapy in patients with positive blood cultures: a randomized controlled trial.</a> Journal of Antimicrobial Chemotherapy. 71 (11), 3276-3283 (2016).</li><li>Baur, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+antibiotic+stewardship+on+the+incidence+of+infection+and+colonisation+with+antibiotic-resistant+bacteria+and+Clostridium+difficile+infection:+a+systematic+review+and+meta-analysis.">Effect of antibiotic stewardship on the incidence of infection and colonisation with antibiotic-resistant bacteria and Clostridium difficile infection: a systematic review and meta-analysis.</a> Lancet Infectious Diseases. 17 (9), 990-1001 (2017).</li><li>Roberts, J. A., Norris, R., Paterson, D. L., Martin, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+drug+monitoring+of+antimicrobials.">Therapeutic drug monitoring of antimicrobials.</a> British Journal of Clinical Pharmacology. 73 (1), 27-36 (2012).</li><li>Paal, M., Zoller, M., Schuster, C., Vogeser, M., Schutze, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+quantification+of+cefepime,+meropenem,+ciprofloxacin,+moxifloxacin,+linezolid+and+piperacillin+in+human+serum+using+an+isotope-dilution+HPLC-MS/MS+method.">Simultaneous quantification of cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid and piperacillin in human serum using an isotope-dilution HPLC-MS/MS method.</a> Journal of Pharmaceutical and Biomedical Analysis. 152, 102-110 (2018).</li><li>Vogeser, M., Seger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pitfalls+associated+with+the+use+of+liquid+chromatography-tandem+mass+spectrometry+in+the+clinical+laboratory.">Pitfalls associated with the use of liquid chromatography-tandem mass spectrometry in the clinical laboratory.</a> Clinical Chemistry. 56 (8), 1234-1244 (2010).</li><li>United States Pharmacopeia and National Formulary. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+&#60;621&#62;.">Chapter &#60;621&#62;.</a> CHROMATOGRAPHY (USP 37-NF 32 S1). , 6376-6385 (2014).</li><li>Wong, G., Sime, F. B., Lipman, J., Roberts, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+do+we+use+therapeutic+drug+monitoring+to+improve+outcomes+from+severe+infections+in+critically+ill+patients?.">How do we use therapeutic drug monitoring to improve outcomes from severe infections in critically ill patients?.</a> BMC Infectious Diseases. 14, 288 (2014).</li><li>. Cefepime hydrochloride: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2016/050679s040lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2016/050679s040lbl.pdf</a> (2016)</li><li>. Meropenem: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2008/050706s022lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2008/050706s022lbl.pdf</a> (2006)</li><li>. Ciprofloxacin hydrochloride: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2016/019537s086lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2016/019537s086lbl.pdf</a> (2016)</li><li>. Moxifloxacin hydrochloride: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2010/021277s038lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2010/021277s038lbl.pdf</a> (2010)</li><li>. Linezolid: Highlights of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2012/021130s028lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2012/021130s028lbl.pdf</a> (2011)</li><li>. Piperacillin and Tazobactam: Highlighs of prescribing information Available from: <a target="_blank" href="https://www.accessdata.fda.gov/drugsatfda_docs/Label/2017/050684s88s89s90_050750s37s38s39lbl.pdf">https://www.accessdata.fda.gov/drugsatfda_docs/Label/2017/050684s88s89s90_050750s37s38s39lbl.pdf</a> (2017)</li><li>Zander, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+biobanking+conditions+on+six+antibiotic+substances+in+human+serum+assessed+by+a+novel+evaluation+protocol.">Effects of biobanking conditions on six antibiotic substances in human serum assessed by a novel evaluation protocol.</a> Clinical Chemistry and Laboratory. 54 (2), 265-274 (2016).</li><li>Zander, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+piperacillin,+tazobactam,+cefepime,+meropenem,+ciprofloxacin+and+linezolid+in+serum+using+an+isotope+dilution+UHPLC-MS/MS+method+with+semi-automated+sample+preparation.">Quantification of piperacillin, tazobactam, cefepime, meropenem, ciprofloxacin and linezolid in serum using an isotope dilution UHPLC-MS/MS method with semi-automated sample preparation.</a> Clinical Chemistry and Laboratory. 53 (5), 781-791 (2015).</li></ol>

The authors have nothing to disclose.

In this manuscript, we report the protocol for a simple and robust tandem mass spectrometry-based method for the quantification of frequently used antibiotics in ICU19, namely cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid, and piperacillin14. A spreadsheet accompanies the manuscript for the preparation of antibiotic stock solutions, calibrators, and quality controls, taking into account the purity of the antibiotics and the molecular weight of their counterions. Given that the concentrations of the antibiotics are rather high, their quantification should be no particular challenge from an analytical perspective. Accordingly, we are confident that this protocol is applicable to various MS instrumental platforms. For a method transfer, users are encouraged to quantify the extra-column volume and hold-up volume of their chromatographic system and to adapt the gradient start time accordingly16. During the method set-up, the system should also be evaluated for carry-over and, if necessary, a blank sample must be injected after the highest calibrator and patient samples with high antibiotic concentrations. Users must also consider the possibility of detector saturation that occurs when too many ions enter a tandem mass spectrometer. Relevant detector saturation can be eliminated with smaller injection volumes, a higher analyte dilution during the sample cleanup, and/or a detuning of a target analyte (e.g., downgrading the optimal voltage settings).
Contrary to other methods, the calibration range allows both a quantification of concentrations close to the MIC of susceptible pathogens, as well as peak concentrations (cmax) that are obtained with a bolus administration. The highest Cmax-values for adults are reported in the corresponding professional information sheets on the FDA drug safety database as follows: 163.9 mg/L for cefepime20, 112 mg/L for meropenem21, 4.6 mg/L for ciprofloxacin22, 4.1 mg/L for moxifloxacin23, 21.2 mg/L for linezolid24, and 298 mg/L for piperacillin25. Antibiotic concentration monitoring in the patient&#39;s blood circulation allows a dose adjustment to the susceptibility of the involved pathogens, but the pharmacokinetic area under the curve can also be obtained through multiple blood sampling with the given protocol.
Many antibiotics (especially beta-lactam meropenem) are chemically unstable once dissolved. The most critical step in this protocol is, therefore, the preparation of the stock solutions, calibrators, and quality controls under cold conditions26,27. In that respect, it is also essential to freeze patient samples as quickly as possible. Although serum storage at -80 &#176;C is recommended26, our stability experiments show that samples can also be stored up to 3 days at -20 &#176;C without any significant decrease of antibiotics concentrations (even at the trough levels).
We recommend performing a system suitability test before each HPLC-MS/MS analysis of patient samples (e.g., with calibrator 3). Generally, a system suitability test is used to verify the repeatability of the LC-MS/MS system and to see if it is also adequate for the analysis to be done. Thus, for instance, decreasing signal intensities are caused by a contamination of the MS sweep cone, which, then, requires its cleaning with an organic solvent. To keep the MS source clean, a divert valve can be introduced after the chromatography column, directing &#34;analyte-free&#34; portions of the mobile phase to the waste before they reach the mass spectrometer. On the other hand, an overall increase of the pressure can indicate column clogging over time. To increase the column longevity usage of a cost-effective precolumn filter is recommendable. If the pressure still continues to be a problem, a flow rate of 0.4 mL/min can also be used with the chromatographic gradient in this protocol.
A minor limitation of this technique is that it requires three separate manual steps for sample clean-up, resulting in a total turnaround time of approximately 30 min. Adding the isotope-labeled internal standards to the precipitation agent may save some processing time. However, this should only be done for high sample throughput rates and with the precipitation agent being stored in the cold (e.g., at -20 &#176;C), as the internal standards also degrade in vitro at elevated temperatures.
The described protocol has been developed for sample processing in standard 1.5 mL polypropylene tubes. Should a higher throughput rate be required for antibiotic TDM, the procedure can be upgraded to the multi-well plate format using adequate centrifuge inserts or filter plates with a vacuum manifold.

Although antibiotics have revolutionized the practice of medicine, severe bacterial infections remain a leading cause of morbidity and mortality in critical illnesses1. In this regard, the prompt administration of a suitable anti-infective in an adequate dosage is of the uppermost importance for disease control2.
A growing body of evidence demonstrates that the empirical treatment with broad-spectrum antibiotics is becoming increasingly problematic with the complexity of patient populations. This is especially true for intensive care units (ICU), where a tremendous inter-individual variability of key pharmacokinetic (PK) parameters is frequently observed3,4. Accordingly, ICU patients are at imminent risk of sub-therapeutic levels with the danger of an insufficient therapeutic success5,6. Then again, patients are unnecessarily exposed to excessively high antibiotic concentrations that may result in serious adverse events with no clinical benefits7. Both the antibiotic misuse and the insufficient dosing have also fueled the dissemination of antibiotic resistance, which is becoming an ever-growing threat to public health8.
To improve the use of antibiotics and to preserve their effectivenessas long as possible, the World Health Organization has launched a global action plan on antimicrobial resistance in 20159. Antibiotic stewardship programs constitute an essential cornerstone of prudent antimicrobial use in national public health strategies10, helping clinicians to improve the quality of patient care11 and, at the same time, significantly reducing the antibiotic resistance12. Antimicrobial dosing in individual patients through the application of therapeutic drug monitoring (TDM) is a key instrument in this context13.
To date, commercially available TDM assays are only available for the glycopeptide antibiotics and aminoglycosides. The quantification of substances from other classes commonly requires an in-house method development or validation that can be cumbersome. We, therefore, present in detail the protocol for a robust mass spectrometry-based assay that can be used for the quantification of the most relevant antibiotics in ICU within their clinical relevant concentration ranges14. The method was recently established in our mass spectrometry facility and has been applied for the routine TDM in ICU since then. The procedure uses a straightforward and simple analytical setting with a uniform sample cleanup, allowing for the rapid implementation of antibiotic TDM in many facilities with mass spectrometry capabilities.
The protocol described here was optimized for the quantification of cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid, and piperacillin in human serum, using isotope dilution liquid chromatography (LC) in combination with a tandem mass spectrometry (MS/MS). For the isotope dilution LC-MS/MS methodology, stable isotope-labeled compounds are added to a sample of interest with a specific matrix (e.g., serum). Isotope-labeled standards can be distinguished from their unlabeled counterpart, namely the analyte of interest, due to different molecular weights of the natural molecule and their fragmentation products, termed a parent-ion-to-daughter-ion transition. As isotope-labeled compounds have an almost identical overall physicochemical behavior compared to their unlabeled counterpart, they are ideal internal standards for the MS/MS, allowing a nearly matrix-independent analyte quantification with a high degree of accuracy15. Nowadays, many stable isotope-labeled internal standards that can be used for small-molecule quantification, including the TDM of antimicrobials, are commercially available.
The chromatographic separation of the antibiotic analytes in the described protocol is performed with an analytical C8 alkyl-chain-length reverse-phase column (100 mm x 2.1 mm, 3 &#181;m particle-size). During the method development, the internal standard normalized matrix factors for all analytes was between 94.6% and 105.4%, with a coefficient of variation of &#8804;8.3%14.

<table><tbody><tr><td>cefepime hydrochloride</td><td>Sigma-Aldrich</td><td>1097636</td><td>USP Reference Standard</td></tr><tr><td>meropenem trihydrate</td><td>Sigma-Aldrich</td><td>Y0001252</td><td>EP Reference Standard</td></tr><tr><td>ciprofloxacin</td><td>Sigma-Aldrich</td><td>17850</td><td></td></tr><tr><td>moxifloxacin hydrochloride</td><td>Sigma-Aldrich</td><td>SML1581</td><td></td></tr><tr><td>linezolid</td><td>Toronto Research Chemicals</td><td>L466500</td><td></td></tr><tr><td>piperacillin sodium salt</td><td>Sigma-Aldrich</td><td>93129</td><td></td></tr><tr><td>cefepime-13C12D3 sulfate</td><td>Alsachim</td><td>C1297</td><td>Isotope labelled internal standard for cefepime</td></tr><tr><td>meropenem-D6</td><td>Toronto Research Chemicals</td><td>M225617</td><td>Isotope labelled internal standard for meropenem</td></tr><tr><td>ciprofloxacin-D8</td><td>Toronto Research Chemicals</td><td>C482501</td><td>Isotope labelled internal standard for ciprofloxacin</td></tr><tr><td>moxifloxacin-13C1D3 hydrochloride</td><td>Toronto Research Chemicals</td><td>M745003</td><td>Isotope labelled internal standard for moxifloxacin</td></tr><tr><td>linezolid-D3</td><td>Toronto Research Chemicals</td><td>L466502</td><td>Isotope labelled internal standard for linezolid</td></tr><tr><td>piperacillin-D5</td><td>Toronto Research Chemicals</td><td>P479952</td><td>Isotope labelled internal standard for piperacillin</td></tr><tr><td>methanol</td><td>JT Baker</td><td>8402</td><td></td></tr><tr><td>HPLC grade water</td><td>JT Baker</td><td>4218</td><td></td></tr><tr><td>formic acid</td><td>Biosolve</td><td>6914132</td><td></td></tr><tr><td>acetic acid</td><td>Biosolve</td><td>1070501</td><td></td></tr><tr><td>ammonium formate</td><td>Sigma-Aldrich</td><td>70221-25G-F</td><td></td></tr><tr><td>tert-Butyl methyl ether</td><td>Merck</td><td>101845</td><td></td></tr><tr><td>Fortis 3 &amp;mu;m C8 100 * 2.1 mm</td><td>Fortis</td><td>F08-020503</td><td></td></tr><tr><td>Ti-PEEK-encased Prifilter (2 &amp;mu;m)</td><td>Chromsystems</td><td>15011</td><td></td></tr><tr><td>2795 Alliance HPLC system</td><td>Waters</td><td>176000491</td><td></td></tr><tr><td>Quattro micro API Tandem Quadrupole System</td><td>Waters</td><td>720000338</td><td></td></tr><tr><td>QuanLynx 4.1 software</td><td>Waters</td><td>/</td><td>Data evaluation software provided by the mass spectrometer manufacturer</td></tr><tr><td>Supplemental File</td><td></td><td></td><td>Stock Solutions</td></tr></tbody></table>

multiplex therapeutic drug monitoring by isotope-dilution hplc-ms/ms of antibiotics in critical illnesses

There is an ever-increasing demand for the therapeutic drug monitoring of antibiotics in many clinical facilities, particularly with regard to the implementation of hospital antibiotic stewardship programs.
In the current work, we present a multiplex high-performance liquid chromatography-tandem mass spectrometry (HPCL-MS/MS) protocol for the quantification of cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid, and piperacillin, commonly used antibiotics in intensive care units. The method was previously comprehensively validated according to the guideline of the European Medicines Agency.
After a rapid sample cleanup, the analytes are separated on a C8 reverse-phase HPLC column within 4 minutes and quantified with the corresponding stable isotope-labeled internal standards in electrospray ionization (ESI+) mass spectrometry in multiple reaction time monitoring (MRM). The presented method uses a simple instrumentation setting with uniform chromatographic conditions, allowing for the daily and robust antibiotic therapeutic drug monitoring in clinical laboratories. The calibration curve spans the pharmacokinetic concentration range, thereby including antibiotic amounts close to the minimal inhibitory concentration (MIC) of susceptible bacteria and peak concentrations (Cmax) that are obtained with bolus administration regimens. Without the necessity of the serum dilution before the sample cleanup, the area under the curve for an administered antibiotic can be obtained through multiple measurements.

Using the described protocol, a typical chromatogram is depicted in Figure 2. According to the United States Pharmacopeia (USP) chromatography guidelines16, the column dead volume in the present system was determined with ~0.22 mL and the extra-column volume (including the injector, tubing, and connectors) with ~0.08 mL, giving a hold-up volume of ~0.30 mL. The calculated retention factors for all analytes were 2.8 (for cefepime) - 4.2 (for piperacillin).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58148/58148fig2.jpg" /> 
Figure 2: Typical analytical chromatogram with normalized signal intensities. The antibiotics are eluting in the following order: cefepime (green), meropenem (brown), ciprofloxacin (red), moxifloxacin (black), linezolid (orange), and piperacillin (purple). The retention times, which are given in minutes, and the analyte peak symmetries vary, depending on the exact composition of the mobile phases, the flow-rate, the chromatography tubing, and the analytical column age. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58148/58148fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 3A contains a sample chart list for the processed samples, including the calibrators 0 - 7 (&#34;Kalibrator 0&#34; - &#34;Kalibrator 7&#34;), quality controls, and patient sera, that are indicated with the injection number (#); the sample identification text (Sample Text); the measured concentration in mg/L (Conc.); the sample type that is either a blank, standard, quality control, or patient sample (Type); the nominal concentration of the calibrators in mg/L (Std. Conc); the analytical retention time (RT); the response that is the ratio of the peak area of the analyte/peak area IS (Response); the deviation from the nominal concentration value (%Dev); the vial position (Vial); and the acquisition time (Acq.Time). The key parameter used for the quantification is the Response, gradually increasing with the analyte concentration, due to the constant amount of added isotope-labeled internal standard.
Figure 3B shows the calibration curve. In regression, the coefficient of determination r2 should be &#62; 0.995. The following calibration model is used for all analytes described in this method: curve type = linear; origin = included; weighting = 1/x; axis transformation = none. In the given example, the calibration curve and quality controls fulfill all quality criteria: r2 &#62; 0.995 for the calibration curve and the deviation of the calibrators (including the LLOQ) and the QC samples is within &#177; 15% of the nominal value.
The measured parent-to-daughter ion transitions (MRM) are given in Figure 3C, showing four peaks at the same retention time: the two upper peaks depict two transitions that are measured for the analyte of interest, the lower two peaks represent the transitions for the corresponding isotope-labeled internal standard. For the quality assessment, the analyte peaks in the respective retention time windows are visually checked and manually reintegrated at the baseline, when necessary.
The minimally inhibitory concentration (MIC) is the central component of the antimicrobial TDM, defining the pharmacokinetic exposure that is required to achieve a target pharmacokinetic/pharmacodynamic (PK/KD) ratio13,17. Accordingly, the target antibiotic TDM concentration levels are expressed in relation to the MIC of the causative pathogen. Given that the action of beta-lactam antibiotics is time-dependent, their efficacy is maximized through the achievement of the therapeutic concentrations that exceed the MIC 4x -5x (fT &#62; 4-5x MIC).When facing unknown infectious pathogens, the target trough concentration range of free (protein-unbound) piperacillin is, therefore, 64 mg/L, corresponding to approximately 90 mg/L total piperacillin18.
The first patient (sample #11) has a satisfactory high serum trough level of 83.4 mg/L piperacillin that is also sufficient for problem pathogens, such as Pseudomonas aeruginosa. The second patient (sample #12) has a concentration of approximately 0.2 mg/L, which is below the lowest calibrator (LLOQ). Perhaps the patient has recovered, and the administration of piperacillin was discontinued. The result &#34;&#60; 0.5 mg/L&#34; is, therefore, reported in the hospital information system. The third patient (sample #13) has a low piperacillin trough concentration of only 5.3 mg/L that is not sufficient for the clear majority of pathogens. For effective antimicrobial chemotherapy, the dosage should be increased by the physician.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58148/58148fig3.jpg" /> 
Figure 3: Exemplary quality assessment and quantification for the analyte piperacillin. These panels represent the mass spectrometry data analysis. (A) This panel shows the sample list, including the calibrators (Standard, samples #1 - #8), quality controls (QC, samples #9 and #10), and patient sera (samples #11 - #13). Calibrator 0 refers to the blank without analyte, but with the addition of an internal standard. 9951 represents QC B, 9953 represents QC D. (B) This panel shows the calibration curve for piperacillin. The percentage deviations from the nominal calibrator concentrations are given in the upper graph (y-axis: residual), the lower graph depicts the linear calibration range. (C) This panel shows the multiple reaction time monitoring (MRM) for piperacillin and the corresponding internal standard piperacillin-D5 for patient serum sample #12. Two parent-to-daughter ion transitions are presented with their retention time and respective signal intensities. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58148/58148fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58148/Supplemental_File-Stock_solutions_04062018.xlsx" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Multiplex Therapeutic Drug Monitoring by Isotope-dilution HPLC-MS/MS of Antibiotics in Critical Illnesses at JoVE.com

Multiplex Therapeutic Drug Monitoring by Isotope-dilution HPLC-MS/MS of Antibiotics in Critical Illnesses

Here we present a tandem mass spectrometry-based protocol for the quantification of frequently used antibiotics in intensive care units, namely cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid, and piperacillin.

There is an ever-increasing demand for the therapeutic drug monitoring of antibiotics in many clinical facilities, particularly with regard to the ...

multiplex-therapeutic-drug-monitoring-isotope-dilution-hplc-msms

Nanyang Technological University

Research

JoVE Journal

Medicine

12.8K Views.  LMU Munich. Hier presenteren we een tandem massa spectrometrie-protocol voor de kwantificering van vaak gebruikte antibiotica in de intensieve zorg eenheden, namelijk cefepime, meropenem ciprofloxacine, moxifloxacin, linezolid en piperacillin.

Video: Multiplex Therapeutic Drug Monitoring by Isotope-dilution HPLC-MS/MS of Antibiotics in Critical Illnesses

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Chemistry

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its therapeutic benefit. Nanoparticle-based drug combination has been shown to mitigate the problems encountered by free drug combination therapy. Our previous studies have shown that the combination of two anticancer drugs, doxorubicin (DOX) and mitomycin C (MMC), produced a synergistic effect against both murine and human breast cancer cells in vitro. DOX and MMC co-loaded polymer-lipid hybrid nanoparticles (DMPLN) bypassed various efflux transporter pumps that confer multidrug resistance and demonstrated enhanced efficacy in breast tumor models. Compared to conventional solution forms, such superior efficacy of DMPLN was attributed to the synchronized pharmacokinetics of DOX and MMC and increased intracellular drug bioavailability within tumor cells enabled by the nanocarrier PLN. 
To evaluate the pharmacokinetics and bio-distribution of co-administered DOX and MMC in both free solution and nanoparticle forms, a simple and efficient multi-drug analysis method using reverse-phase high performance liquid chromatography (HPLC) was developed. In contrast to previously reported methods that analyzed DOX or MMC individually in the plasma, this new HPLC method is able to simultaneously quantitate DOX, MMC and a major cardio-toxic DOX metabolite, doxorubicinol (DOXol), in various biological matrices (e.g., whole blood, breast tumor, and heart). A dual fluorescent and ultraviolet absorbent probe 4-methylumbelliferone (4-MU) was used as an internal standard (I.S.) for one-step detection of multiple drug analysis with different detection wavelengths. This method was successfully applied to determine the concentrations of DOX and MMC delivered by both nanoparticle and solution approaches in whole blood and various tissues in an orthotopic breast tumor murine model. The analytical method presented is a useful tool for pre-clinical analysis of nanoparticle-based delivery of drug combinations.

This protocol describes an efficient and convenient analytical process of sample extraction and simultaneous determination of multiple drugs, doxorubicin (DOX), mitomycin C (MMC) and a cardio-toxic DOX metabolite, doxorubicinol (DOXol), in the biological samples from a preclinical breast tumor model treated with nanoparticle formulations of synergistic drug combination.

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its ...

Cancer Research

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies that allow simultaneous measurement of multiple samples have become widespread and greatly reduce experimental costs and times. Our laboratory developed a technique called combined precursor isotopic labeling and isobaric tagging (cPILOT), which enhances sample multiplexing of traditional isotopic labeling or isobaric tagging approaches. Global cPILOT can be applied to samples originating from cells, tissues, bodily fluids, or whole organisms and gives information on relative protein abundances across different sample conditions. cPILOT works by 1) using low pH buffer conditions to selectively dimethylate peptide N-termini and 2) using high pH buffer conditions to label primary amines of lysine residues with commercially-available isobaric reagents (see Table of Materials/Reagents). The degree of sample multiplexing available is dependent on the number of precursor labels used and the isobaric tagging reagent. Here, we present a 12-plex analysis using light and heavy dimethylation combined with six-plex isobaric reagents to analyze 12 samples from mouse tissues in a single analysis. Enhanced multiplexing is helpful for reducing experimental time and cost and more importantly, allowing comparison across many sample conditions (biological replicates, disease stage, drug treatments, genotypes, or longitudinal time-points) with less experimental bias and error. In this work, the global cPILOT approach is used to analyze brain, heart, and liver tissues across biological replicates from an Alzheimer&#39;s disease mouse model and wild-type controls. Global cPILOT can be applied to study other biological processes and adapted to increase sample multiplexing to greater than 20 samples.

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is a quantitative proteomics strategy that enhances sample multiplexing capabilities of isobaric tags. This protocol describes the application of cPILOT to tissues from an Alzheimer's disease mouse model and wild-type controls.

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies ...

Biochemistry

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Bioengineering

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 ...

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 ...

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of multiplexing up to 22 or 24 samples with tandem mass tags or isobaric N,N-dimethyl leucine isobaric tags, respectively, in a single experiment. This enhanced sample multiplexing considerably reduces mass spectrometry acquisition times and increases the utility of the expensive commercial isobaric reagents. However, the manual process of sample handling and pipetting steps in the strategy can be labor intensive, time consuming, and introduce sample loss and quantitative error. These limitations can be overcome through the incorporation of automation. Here we transferred the manual cPILOT protocol to an automated liquid handling device that can prepare large sample numbers (i.e., 96 samples) in parallel. Overall, automation increases feasibility and reproducibility of cPILOT and allows for broad usage by other researchers with comparable automation devices.

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is an enhanced sample multiplexing strategy that is capable of increasing the number of samples that can be analyzed simultaneously with available isobaric tags. Incorporation of a robotic platform has greatly increased experimental throughput, reproducibility, and quantitative accuracy.

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of ...

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 ...

Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) is the most widely used method in qualitative analysis of compounds. The method includes standardized and programmed protocols for sample pretreatment, MS tune, MS acquisition, and data processing. The sample pretreatments include collection, pulverization, solvent extraction, ultrasound, centrifugation, and filtration. Data post-processing was described in detail and includes data importing, self-established database construction, method establishment, data processing, and other manual operations. The above-ground part of the alpine yarrow herb, Achillea millefolium L., is used to treat inflammation, gastrointestinal disturbances, and pain and its 3-oxa-guaianolides could be useful leads for anti-inflammatory drug development. Three representative compounds in AML were identified, combining TOF-MS with a self-established database. Moreover, the differences from existing literature, liquid-phase parameter optimization, scan mode selection, ion source suitability, collision energy adjustment, isomer screening, method limitation, and possible solutions were discussed. This standardized analysis method is universal and can be applied to identify complex compounds in Chinese herbal medicine.

We describe a general protocol and systematic design that could be applied to separate and recognize complex components in alpine yarrow herb, Achillea millefolium L., a Chinese herbal medicine.

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography ...

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.

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. ...

Multiplex Therapeutic Drug Monitoring by Isotope-dilution HPLC-MS/MS of Antibiotics in Critical Illnesses

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video