Editing High-performance liquid chromatography (section)

== Applications ==

=== Manufacturing ===
HPLC has many applications in both laboratory and clinical science. It is a common technique used in pharmaceutical development, as it is a dependable way to obtain and ensure product purity.<ref>{{cite journal  |title=Practical aspects of fast reversed-phase high-performance liquid chromatography using 3 μm particle packed columns and monolithic columns in pharmaceutical development and production working under current good manufacturing practice |last1=Gerber |first1=Frederic |date=May 2004 |journal=Journal of Chromatography |volume=1036 |issue=2 |pages=127–33 |doi=10.1016/j.chroma.2004.02.056 |pmid=15146913 }}</ref> While HPLC can produce extremely high quality (pure) products, it is not always the primary method used in the production of bulk drug materials.<ref>{{cite journal |title=Analytical techniques in pharmaceutical analysis: A review |journal=Arabian Journal of Chemistry |volume=10 |pages=S1409–S1421 |doi=10.1016/j.arabjc.2013.04.016 |first1=Masoom Raza |last1=Siddiqui |first2=Zeid A. |last2=AlOthman |first3=Nafisur |last3=Rahman|year=2013 |doi-access=free }}</ref> According to the European pharmacopoeia, HPLC is used in only 15.5% of syntheses.<ref>''The European Pharmacopoeia'', 2002. fourth ed., Council of Europe, Strasbourg.</ref> However, it plays a role in 44% of syntheses in the United States pharmacopoeia.<ref>''United States Pharmacopoeia'', 2004. 27th ed. The USP Convention Inc., Rockville, MD.</ref> This could possibly be due to differences in monetary and time constraints, as HPLC on a large scale can be an expensive technique. An increase in specificity, precision, and accuracy that occurs with HPLC unfortunately corresponds to an increase in cost.

=== Legal ===
This technique is also used for detection of [[Prohibition of drugs|illicit drugs]] in various samples.<ref>{{Cite journal |last1=Merone |first1=Giuseppe M. |last2=Tartaglia |first2=Angela |last3=Rossi |first3=Sandra |last4=Santavenere |first4=Francesco |last5=Bassotti |first5=Elisa |last6=D'Ovidio |first6=Cristian |last7=Bonelli |first7=Martina |last8=Rosato |first8=Enrica |last9=de Grazia |first9=Ugo |last10=Locatelli |first10=Marcello |last11=Savini |first11=Fabio |date=2021 |title=Fast Quantitative LC-MS/MS Determination of Illicit Substances in Solid and Liquid Unknown Seized Samples |journal=Analytical Chemistry |language=en |volume=93 |issue=49 |pages=16308–16313 |doi=10.1021/acs.analchem.1c03310 |issn=0003-2700 |pmc=8674870 |pmid=34843645}}</ref> The most common method of drug detection has been an [[immunoassay]].<ref>{{Cite journal|title = An evaluation of the diagnostic accuracy of liquid chromatography-tandem mass spectrometry versus immunoassay drug testing in pain patients|url=http://www.painphysicianjournal.com/current/pdf?article=MTM1MQ%3D%3D&journal=55|journal = Pain Physician|date = 2010-06-01|pmid = 20495592|pages = 273–281|volume = 13|issue = 3|first1 = Amadeo|last1 = Pesce|first2 = Murray|last2 = Rosenthal|first3 = Robert|last3 = West|first4 = Cameron|last4 = West|first5 = Bridgit|last5 = Crews|first6 = Charles|last6 = Mikel|first7 = Perla|last7 = Almazan|first8 = Sergey|last8 = Latyshev}}</ref> This method is much more convenient. However, convenience comes at the cost of specificity and coverage of a wide range of drugs, therefore, HPLC has been used as well as an alternative method. As HPLC is a method of determining (and possibly increasing) purity, using HPLC alone in evaluating concentrations of drugs was somewhat insufficient. Therefore, HPLC in this context is often performed in conjunction with [[mass spectrometry]].<ref>{{Cite journal|title = Screening and confirmation of 62 drugs of abuse and metabolites in urine by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry|journal = Journal of Analytical Toxicology|date = 2013-12-01|pmid = 24084874|pages = 642–651|volume = 37|issue = 9|doi = 10.1093/jat/bkt083|first1 = I.-Lin|last1 = Tsai|first2 = Te-I.|last2 = Weng|first3 = Yufeng J.|last3 = Tseng|first4 = Happy Kuy-Lok|last4 = Tan|first5 = Hsiao-Ju|last5 = Sun|first6 = Ching-Hua|last6 = Kuo|doi-access = free}}</ref> Using [[Liquid chromatography–mass spectrometry|liquid chromatography-mass spectrometry]] (LC-MS) instead of [[Gas chromatography–mass spectrometry|gas chromatography-mass spectrometry]] (GC-MS) circumvents the necessity for derivitizing with acetylating or alkylation agents, which can be a burdensome extra step.<ref>{{Cite journal|title = Automated solid-phase extraction and two-step derivatisation for simultaneous analysis of basic illicit drugs in serum by GC/MS|journal = International Journal of Legal Medicine|date = 2000-01-01|pmid = 10929239|pages = 229–235|volume = 113|issue = 4|first1 = W.|last1 = Weinmann|first2 = M.|last2 = Renz|first3 = S.|last3 = Vogt|first4 = S.|last4 = Pollak|doi=10.1007/s004149900098|s2cid = 20451772}}</ref> LC-MS has been used to detect a variety of agents like doping agents, drug metabolites, glucuronide conjugates, amphetamines, opioids, cocaine, BZDs, ketamine, LSD, cannabis, and pesticides.<ref>{{Cite journal|title = A general screening method for doping agents in human urine by solid phase extraction and liquid chromatography/time-of-flight mass spectrometry|journal = Analytica Chimica Acta|date = 2007-02-28|pmid = 17386652|pages = 94–102|volume = 585|issue = 1|doi = 10.1016/j.aca.2006.12.028|first1 = Marjo|last1 = Kolmonen|first2 = Antti|last2 = Leinonen|first3 = Anna|last3 = Pelander|first4 = Ilkka|last4 = Ojanperä| bibcode=2007AcAC..585...94K }}</ref><ref>{{Cite journal|title = Toxicological screening with formula-based metabolite identification by liquid chromatography/time-of-flight mass spectrometry|journal = Analytical Chemistry|date = 2003-11-01|pmid = 14588010|pages = 5710–5718|volume = 75|issue = 21|doi = 10.1021/ac030162o|first1 = Anna|last1 = Pelander|first2 = Ilkka|last2 = Ojanperä|first3 = Suvi|last3 = Laks|first4 = Ilpo|last4 = Rasanen|first5 = Erkki|last5 = Vuori}}</ref> Performing HPLC in conjunction with mass spectrometry reduces the absolute need for standardizing HPLC experimental runs.

=== Research ===
Similar assays can be performed for research purposes, detecting concentrations of potential clinical candidates like anti-fungal and asthma drugs.<ref>{{Cite journal|title = Metabolic profiling of a potential antifungal drug, 3-(4-bromophenyl)-5-acetoxymethyl-2,5-dihydrofuran-2-one, in mouse urine using high-performance liquid chromatography with UV photodiode-array and mass spectrometric detection|journal = Journal of Chromatography B|date = 2007-06-15|pmid = 17400036|pages = 10–19|volume = 853|issue = 1–2|doi = 10.1016/j.jchromb.2007.02.045|first1 = Milan|last1 = Nobilis|first2 = Milan|last2 = Pour|first3 = Petr|last3 = Senel|first4 = Jan|last4 = Pavlík|first5 = Jirí|last5 = Kunes|first6 = Marie|last6 = Voprsalová|first7 = Lenka|last7 = Kolárová|first8 = Michal|last8 = Holcapek}}</ref> This technique is obviously useful in observing multiple species in collected samples, as well, but requires the use of standard solutions when information about species identity is sought out. It is used as a method to confirm results of synthesis reactions, as purity is essential in this type of research. However, mass spectrometry is still the more reliable way to identify species.

=== Medical and health sciences ===
Medical use of HPLC typically use mass spectrometer (MS) as the detector, so the technique is called LC-MS<ref>{{Cite web |last1=Gu |first1=Jatin |last2=Patel |first2=Kumar |last3=Shah |first3=Dhiren |date=2016 |title=APPLICATION OF LC-MS |url=https://www.pharmatutor.org/articles/application-liquid-chromatography-mass-spectrometry |website=PharmaTutor |language=en}}</ref> or LC-MS/MS for tandem MS, where two types of MS are operated sequentially.<ref>{{Cite journal |last1=Tallam |first1=Anil Kumar |last2=Alapati |first2=Sahithi |last3=Nuli |first3=Mohana Vamsi |date=2023 |title=A review on bioanalytical method development and validation of anticancer drugs by using lc/ms/ms and its applications on routine analysis |url=https://www.jisciences.com/index.php/journal/article/view/51 |journal=Journal of Integral Sciences |language=en |pages=4–19 |doi=10.37022/jis.v6i1.51 |s2cid=257295079 |issn=2581-5679|doi-access=free }}</ref> When the HPLC instrument is connected to more than one detector, it is called a hyphenated LC system.{{citation needed|date=February 2024}} Pharmaceutical applications<ref>{{Cite web |last=O'Driscoll |first=Aimee |date=2021 |title=HPLC in Pharmaceutical Applications |url=https://www.labmanager.com/hplc-in-pharmaceutical-applications-25323 |website=Lab Manager |language=en}}</ref> are the major users of HPLC, LC-MS and LC-MS/MS.<ref>{{Cite journal |last1=Beccaria |first1=Marco |last2=Cabooter |first2=Deirdre |date=2020 |title=Current developments in LC-MS for pharmaceutical analysis |url=https://pubs.rsc.org/en/content/articlelanding/2020/an/c9an02145k |journal=Analyst |language=en |volume=145 |issue=4 |pages=1129–1157 |doi=10.1039/C9AN02145K |pmid=31971527 |bibcode=2020Ana...145.1129B |hdl=11392/2479221 |s2cid=210866236 |issn=1364-5528|hdl-access=free }}</ref> This includes drug development<ref>{{Cite journal |last1=Gu |first1=Chunang (Christine) |last2=Russell |first2=David |last3=Yehl |first3=Peter |date=2016 |title=Application of LCMS in small-molecule drug development |url=https://www.europeanpharmaceuticalreview.com/article/43628/application-lcms-small-molecule-drug-development/ |journal=European Pharmaceutical Review |language=en |volume=21 |issue=4 |pages=54–57}}</ref> and pharmacology, which is the scientific study of the effects of drugs and chemicals on living organisms,<ref>{{Cite journal |last1=D'Ovidio |first1=Cristian |last2=Locatelli |first2=Marcello |last3=Perrucci |first3=Miryam |last4=Ciriolo |first4=Luigi |last5=Furton |first5=Kenneth G. |last6=Gazioglu |first6=Isil |last7=Kabir |first7=Abuzar |last8=Merone |first8=Giuseppe Maria |last9=de Grazia |first9=Ugo |last10=Ali |first10=Imran |last11=Catena |first11=Antonio Maria |last12=Treglia |first12=Michele |last13=Marsella |first13=Luigi T. |last14=Savini |first14=Fabio |date=2023 |title=LC-MS/MS Application in Pharmacotoxicological Field: Current State and New Applications |journal=Molecules |language=en |volume=28 |issue=5 |pages=2127 |doi=10.3390/molecules28052127 |issn=1420-3049 |pmc=10004468 |pmid=36903374 |doi-access=free }}</ref> personalized medicine,<ref>{{Cite journal |last1=Zhou |first1=Juntuo |last2=Zhong |first2=Lijun |date=2022 |title=Applications of liquid chromatography-mass spectrometry based metabolomics in predictive and personalized medicine |journal=Frontiers in Molecular Biosciences |volume=9 |doi=10.3389/fmolb.2022.1049016 |issn=2296-889X |pmc=9669074 |pmid=36406271 |doi-access=free }}</ref> public health<ref>{{Cite journal |last1=Mathias |first1=Patricia I. |last2=Connor |first2=Thomas H. |last3=B'Hymer |first3=Clayton |date=2017 |title=A review of high performance liquid chromatographic-mass spectrometric urinary methods for anticancer drug exposure of health care workers |journal=Journal of Chromatography B |volume=1060 |pages=316–324 |doi=10.1016/j.jchromb.2017.06.028 |issn=1570-0232 |pmc=5585056 |pmid=28654869}}</ref><ref>{{Cite journal |last1=Hernández |first1=Félix |last2=Sancho |first2=Juan V. |last3=Ibáñez |first3=María |last4=Guerrero |first4=Carlos |date=2007 |title=Antibiotic residue determination in environmental waters by LC-MS |url=https://www.sciencedirect.com/science/article/pii/S0165993607000295 |journal=TrAC Trends in Analytical Chemistry |series=Pharmaceutical-residue analysis |volume=26 |issue=6 |pages=466–485 |doi=10.1016/j.trac.2007.01.012 |issn=0165-9936}}</ref> and diagnostics.<ref>{{Cite journal |last1=Seger |first1=Christoph |last2=Salzmann |first2=Linda |date=2020 |title=After another decade: LC–MS/MS became routine in clinical diagnostics |journal=Clinical Biochemistry |series=Advancement and Applications of Mass Spectrometry in Laboratory Medicine |volume=82 |pages=2–11 |doi=10.1016/j.clinbiochem.2020.03.004 |pmid=32188572 |s2cid=213186669 |issn=0009-9120|doi-access=free }}</ref> While urine is the most common medium for analyzing drug concentrations, blood serum is the sample collected for most medical analyses with HPLC.<ref>{{Cite journal|title = A high-sensitivity ultra-high performance liquid chromatography/high-resolution time-of-flight mass spectrometry (UHPLC-HR-TOFMS) method for screening synthetic cannabinoids and other drugs of abuse in urine|journal = Analytical and Bioanalytical Chemistry|date = 2013-10-01|pmid = 23954996|pages = 8463–8474|volume = 405|issue = 26|doi = 10.1007/s00216-013-7272-8|first1 = Mira|last1 = Sundström|first2 = Anna|last2 = Pelander|first3 = Verena|last3 = Angerer|first4 = Melanie|last4 = Hutter|first5 = Stefan|last5 = Kneisel|first6 = Ilkka|last6 = Ojanperä|s2cid = 25743579}}</ref> One of the most important roles of LC-MS and LC-MS/MS in the clinical lab is the Newborn Screening (NBS) for metabolic disorders<ref>{{Cite journal |last1=Gelb |first1=Michael H. |last2=Basheeruddin |first2=Khaja |last3=Burlina |first3=Alberto |last4=Chen |first4=Hsiao-Jan |last5=Chien |first5=Yin-Hsiu |last6=Dizikes |first6=George |last7=Dorley |first7=Christine |last8=Giugliani |first8=Roberto |last9=Hietala |first9=Amy |last10=Hong |first10=Xinying |last11=Kao |first11=Shu-Min |last12=Khaledi |first12=Hamid |last13=Klug |first13=Tracy |last14=Kubaski |first14=Francyne |last15=Liao |first15=Hsuan-Chieh |date=2022 |title=Liquid Chromatography–Tandem Mass Spectrometry in Newborn Screening Laboratories |journal=International Journal of Neonatal Screening |language=en |volume=8 |issue=4 |pages=62 |doi=10.3390/ijns8040062 |issn=2409-515X |pmc=9781967 |pmid=36547379 |doi-access=free }}</ref> and follow-up diagnostics.<ref>{{Cite journal |last1=Wang |first1=Yanyun |last2=Sun |first2=Yun |last3=Jiang |first3=Tao |date=2019 |title=Clinical Application of LC–MS/MS in the Follow-Up for Treatment of Children with Methylmalonic Aciduria |url=https://doi.org/10.1007/s12325-019-00955-0 |journal=Advances in Therapy |language=en |volume=36 |issue=6 |pages=1304–1313 |doi=10.1007/s12325-019-00955-0 |pmid=31049874 |s2cid=143432183 |issn=1865-8652}}</ref><ref>{{Cite journal |last1=Arunkumar |first1=Nivethitha |last2=Langan |first2=Thomas J. |last3=Stapleton |first3=Molly |last4=Kubaski |first4=Francyne |last5=Mason |first5=Robert W. |last6=Singh |first6=Rajendra |last7=Kobayashi |first7=Hironori |last8=Yamaguchi |first8=Seiji |last9=Suzuki |first9=Yasuyuki |last10=Orii |first10=Kenji |last11=Orii |first11=Tadao |last12=Fukao |first12=Toshiyuki |last13=Tomatsu |first13=Shunji |date=2020 |title=Newborn screening of mucopolysaccharidoses: past, present, and future |url=https://www.nature.com/articles/s10038-020-0744-8 |journal=Journal of Human Genetics |language=en |volume=65 |issue=7 |pages=557–567 |doi=10.1038/s10038-020-0744-8 |pmid=32277174 |s2cid=92042115 |issn=1435-232X}}</ref> The infants' samples come in the shape of dried blood spot (DBS),<ref>{{Cite journal |last1=Skogvold |first1=Hanne Bendiksen |last2=Rootwelt |first2=Helge |last3=Reubsaet |first3=Léon |last4=Elgstøen |first4=Katja Benedikte Prestø |last5=Wilson |first5=Steven Ray |date=2023 |title=Dried blood spot analysis with liquid chromatography and mass spectrometry: Trends in clinical chemistry |url=https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/jssc.202300210 |journal=Journal of Separation Science |language=en |volume=46 |issue=15 |pages=e2300210 |doi=10.1002/jssc.202300210 |pmid=37269205 |s2cid=259047202 |issn=1615-9306|hdl=10852/105845 |hdl-access=free }}</ref> which is simple to prepare and transport, enabling safe and accessible diagnostics, both locally and globally.

Other methods of detection of molecules that are useful for clinical studies have been tested against HPLC, namely immunoassays. In one example of this, competitive protein binding assays (CPBA) and HPLC were compared for sensitivity in detection of vitamin D. Useful for diagnosing vitamin D deficiencies in children, it was found that sensitivity and specificity of this CPBA reached only 40% and 60%, respectively, of the capacity of HPLC.<ref>{{Cite journal|title = Competitive Protein-binding assay-based Enzyme-immunoassay Method, Compared to High-pressure Liquid Chromatography, Has a Very Lower Diagnostic Value to Detect Vitamin D Deficiency in 9–12 Years Children|journal = International Journal of Preventive Medicine|date = 2015-01-01|pmc = 4542329|pmid = 26330983|page = 67|volume = 6|doi = 10.4103/2008-7802.161069|first1 = Maliheh|last1 = Zahedi Rad|first2 = Tirang Reza|last2 = Neyestani|first3 = Bahareh|last3 = Nikooyeh|first4 = Nastaran|last4 = Shariatzadeh|first5 = Ali|last5 = Kalayi|first6 = Niloufar|last6 = Khalaji|first7 = Azam|last7 = Gharavi | doi-access=free }}</ref> While an expensive tool, the accuracy of HPLC is nearly unparalleled.