June 2019

Medical Mass Spectrometry Vol. 3 No. 1

Review

Applications of mass spectrometry in clinical chemistry
Mamoru Satoh, Fumio Nomura*Division of Clinical Mass Spectrometry, Chiba University Hospital, 1‒8‒1 Inohana, Chuo-ku, Chiba-shi, Chiba 260‒8670, Japan
Abstract

Mass spectrometry (MS) is now an essential technology for laboratory medicine. The most successful applica-tion of MS in laboratory medicine is the rapid identiﬁcation of microorganisms using matrix-assisted laser desorption/ionization time-of-ﬂight (MALDI-TOF) MS. However, the adoption of MS-based assays for routine testing in clinical chemistry is very slow in Japan. In this review, we summarize the current status of clinical mass spectrometry in the US, Europe,
and Japan, and discuss the possible reasons why Japan is behind in this regard.
Liquid chromatography-tandem mass spectrometry (LC/MS/MS) is a highly accurate and reproducible analytical technique, but a number of challenges and pitfalls remain for routine use in clinical laboratories, such as the effects of various
pre-analytical factors. Indeed, we observed a signiﬁcant noise peak during routine measurements of vitamin D metabolites,
most likely due to a separating gel in blood collection tubes used for particular specimens sent by another hospital. Ion suppression due to matrix effects and problems associated with stable isotope labeled internal standards should also be considered.
MS assays are typically laboratory developed tests at present. As LC/MS/MS procedures become more automated andmore MS-related in vitro diagnostics become commercially available, the application of LC/MS/MS to laboratory medicine
will be signiﬁcantly accelerated.
Key words: LC/MS/MS, MALDI-TOF MS, clinical chemistry, matrix effect, pre-analytical factors

Introduction

ics . For example, it was recently reported that the BoxCar
acquisition method enables single-shot proteomics analysis

Advances in mass spectrometer have been remarkable,
and increasing numbers of highly sensitive, user-friendly1)of 10,000 proteins in 100 minutes . Over recent years,

novel instruments are released by instrument manufacturers

sophisticated instruments have been developed not only forevery year. Mass spectrometry (MS) is now an essential
technology for basic biological research and clinical medi-

basic research but also for specialized clinical applications.
In the latter cases, sample preparation and LC-MS are inte-cine. Rapid progress in MS-related technologies, including
sample preparation methods, labeling reagents, stable iso-grated into the system. Moreover, advanced ionization process, operating system, and the required reagents including
calibrators, quality control samples and ready-to-use sol-

tope labeling reagents and peptide synthesis technologies,
has led to the evolution of Proteomics and Metabolom-

vents are now commercially available. Several ionization
methods, such as Desorption Electrospray Ionization*Corresponding author

2)

3)(DESI) , Probe Electro Spray Ionization (PESI)

Fumio Nomura, MD, PhD.

Rapid

Spectrometry(REIMS) have been used for intraoperative rapid pathological diagnosis and real-time analysis of biological sam-Division of Clinical Mass Spectrometry, Chiba University
Hospital, 1‒8‒1 Inohana, Chuo-ku, Chiba-shi, Chiba 260‒

Evaporative

Ionization

and

Mass

4)

8670, Japan
Tel: ＋81‒43‒226‒2627, Fax: ＋81‒43‒226‒2627

ples. Mass cytometry is a novel technique similar to ﬂow
cytometry and is widely used in hematological examina-

E-mail: fnomura@faculty.chiba-u.jp

5)Received March 18, 2019. Accepted April 26, 2019.

tions . Clinical applications of mass spectrometry will

Epub June 18, 2019.

become more widespread as technological innovations

DOI: 10.24508/mms.2019.06.005

advance.
2June 2019

Medical Mass Spectrometry Vol. 3 No. 1

stances, antibodies are not speciﬁc enough and may lead tocross-reactivity between closely related compounds. LC/

MS is used in various ﬁelds in Japan, including newborn
screening for inborn errors of metabolism, forensic toxicology, and therapeutic drug monitoring. The most successful
application of MS in laboratory medicine is the rapid identi-

MS/MS can overcome this limitation and is increasingly

ﬁcation of microorganisms using matrix-assisted laser
desorption/ionization time-of-ﬂight (MALDI-TOF) MS.
Indeed, there has been a revolutionary shift in clinical diag-

United States. For example, approximately 15% of theused for clinical chemistry, mainly in Europe and thepapers presented at the scientiﬁc poster session of theAmerican Association for Clinical Chemistry (AACC) overthe past few years dealt with Mass Spectrometry Applica-

nostic microbiology . Since an early report of the application of MALDI-TOF MS-based bacterial identiﬁcation in
6)

tions. At AACC, a number of MS manufacturers display
and introduce their new instruments for laboratory person-

7)clinical laboratory on a routine basis , this technology isincreasingly used globally. In Japan, as of August 2018,
there were around 200 instruments (either MALDI Bio-nel. The Association for Mass Spectrometry Applications to
the Clinical Labs (MSACL) has the mission to accelerate
the implementation of mass spectrometry in the clinical lab-®

typer: Bruker, USA or VITEK MS: bioMérieux, France)in operation for routine clinical use. MALDI-TOF MS isoratory with the goal of contributing to the improvement ofpatient standard-of-care as well as reducing health careparticularly useful for rapid bacterial identiﬁcation in blood
culture specimens, providing analyses 1.5 to 2 days fasterthan conventional methods and thus helping shorten hospi-

costs. Their annual meetings are held twice a year, one eachtal stays and improve overall prognosis. Moreover, a 2018
revision of the Japanese health insurance medical fee sys-

The Clinical Laboratory Improvement Amendments(CLIA) in the United States is a body of United States fed-

tem allows additional charges for microbial identiﬁcation
by MS-based methods. This development will accelerateeral regulatory standards. CLIA certiﬁed laboratories canin the United States of America and Europe.

the implementation of MALDI-TOF MS in major hospitals

conduct laboratory developed tests (LDT), including
LC-MS/MS, in the absence of ofﬁcially certiﬁed IVDs, andin Japan.

health insurance reimbursement is generally possible. More

In contrast, the adoption of liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assays for rou-

recently, FDA has decided to extend their regulations totine clinical chemistry testing is very slow in Japan andremains limited to large institutions and reference laborato-

unknown. A provocative editorial was published in the

LDTs as well. The details of this policy have remained

ries. Many challenges and limitations prevent LC-MS/MS

Journal of Clinical Endocrinology and Metabolism (JCEM)
8)
in 2013 indicating that effective January 1, 2015, manu-

from being implemented routinely. First, LC/MS/MS
involves complex, labor-intensive workﬂow compared toscripts reporting sex steroid assays as important endpoints
must be MS-based assays instead of immunoassays, and thefully automated immunoassay platforms. Another bottleneck is the absence of ofﬁcially approved in vitro diagnos-

methods used must be reported or cited in sufﬁcient detail.
Although this policy has been suspended because of thecomplexity of the issues, we believe that it will be eventu-

tic reagents (IVD), including calibrators and preparation
reagents, although there is an FDA-cleared test kit (SCIEX
TM

ally effective in the very near future.

System). As more

The situation in Japan: Immunoassays are also widely

IVD reagents and kits are ofﬁcially approved and becomeused in Japan because of the minimal sample preparationcommercially available, more LC/MS/MS platforms will be

required and rapid turnaround time (TAT). Most laboratory
tests for outpatients are conducted as a same-day test in

Vitamin D 200M Assay for the Topazwidely used in clinical chemistry laboratories.

hospitals that have their clinical laboratories, which

Current Status of Mass Spectrometry in Clinical Chemistrydemands short TAT of less than one hour. This requirement
for rapid TAT is one reason preventing the widespread useof LC-MS/MS in the clinical chemistry ﬁeld in Japan.

The situation in the US and Europe: Currently, immuno-

assays are widely used for testing proteins, peptides and

LC/MS/MS can serve as a gold standard for evaluating
the accuracy of other methods. Serum 25-hydroxyvitamin

hormones in clinical laboratories. However, immunoassays

D [25(OH)D] is an indicator of vitamin D status and is gen-have several limitations and drawbacks, such as being
dependent on antibody speciﬁcity. Under many circum-

erally quantiﬁed by immunoassays in Japan. The Japanese3June 2019

Medical Mass Spectrometry Vol. 3 No. 1

between the antibody and other low-abundance metabolites
10)with similar structures . We speculate that a similar expla-

Society of Clinical Chemistry recently evaluated the standardization of six automated immunoassays available in
9)

10)

Japan using the LC/MS/MS method

developed at Chiba

nation will be possible for the discordant results of E2, but

University Hospital as a reference method.

exact reasons remain to be clariﬁed.

The range of correlation coefﬁcients between the values

Challenges and pitfalls in clinical applicationsof LC-MS/MS

obtained by LC/MS/MS and each immunoassay was 0.73
to 0.97. The results obtained using six immunoassay kits
deviated greatly from the MS assay results (−28.6% to

LC-MS/MS is a highly accurate analytical technique

42.2%). The discrepancies in values obtained using the six

increasingly used in clinical chemistry, but a number of

immunoassay kits ranged from 3.7% to 18.1%. The

challenges and pitfalls remain for its routine use in clinical13)
laboratories : speciﬁcally, the effects of various pre-analyt-researchers concluded that current automated 25(OH)D
manufacturers pursue quality assurance so that values

ical factors, ion suppression due to matrix effect, the problems associated with stable isotope labeled internal stan-obtained using their kits align more closely to the values ofdards, and lack of automation and standardization.

immunoassays lack standardization and recommended thatinternationally accepted standard reference materials.
Accurate measurement of plasma aldosterone concentration is a prerequisite for the diagnosis of primary aldostero-Pre-analytical factors

nism and is currently conducted by immunoassays. Nishi-Interferences by bilirubin, hemoglobin, and chyle have tokawa et al. studied the standardization of aldosterone

be checked in LC/MS/MS as well. When antibody use ismeasurements in blood using serum certiﬁed reference
11)material and isotope dilution-liquid chromatography-tan-

included in the ﬁrst step of sample preparation as in the14)case with 1 α ,25-dihydorxyvitamin D measurements ,dem mass spectrometry (ID-LC/MS/MS) as the reference

effects of a rheumatoid factor (FA) to speciﬁc antibody formethod. They reported that the aldosterone values obtained

the analyte have to be excluded. Antibodies are often usedby the representative RIA (radioimmunoassay) using thein targeted proteomics to quantify low abundant peptides

current calibrator did not agree well with those obtainedusing certiﬁed reference material from the National Metrol-(on the order of pg/mL) in blood .

ogy Institute of Japan (NMIJ). Given these results, the Japa-

try labs are designed basically for colorimetric assays andnese Endocrine Society recommended the use of certiﬁed
reference material to ensure the traceability of each immu-

immunoassays and are not supposed to be used for LC/MS/

Quality assurance of IVDs is conducted in many ways.

15)

Blood collection tubes currently used in clinical chemis-

noassay. Undoubtedly, the current immunoassays for aldo-

MS. Therefore, it is possible that unexpected results are
obtained when MS-based assays are carried out using thesesterone should be improved in terms of speciﬁcity, but webelieve that LC/MS/MS should eventually take over immu-

tubes. Wang et al. reported that testosterone levels determined by LC-MS/MS in sera collected in particular tubes

noassays for plasma or serum aldosterone measurements.

gave results 4-fold higher than those collected in the plain
16)

We often observe discrepancies between the values

tubes which do not contain any additives . It was mostobtained by immunoassays and LC/MS/MS in the method

likely that any component of separating gel was responsible

development process. Serum estradiol (E2) levels arefor the interference. We experienced a similar case whenmostly measured by immunoassay in Japan. Although E2

we changed the transition in our LC/MS/MS method for

immunoassays are useful when fast TAT is required, they
are not speciﬁc enough for post-menopausal subjects and inserum 25(OH)D3

the diagnosis of sex hormone-related disorders. We com-peak in clinical specimens provided by Hospital A (Fig.

pared serum E2 levels obtained by LC/MS/MS with thoseby two representative immunoassays available in Japan1a). The noise peak disappeared in the transition before the17)change (Fig. 1b) . This noise was observed in every speci-

and found that both immunoassays exhibited a remarkablemen from Hospital A, but not in specimens collected in our

positive bias, particularly at low E2 levels.

hospital. Although the speciﬁc reason is yet unknown, we

In the case of vitamin D measurements, we have demonstrated that the discrepancy resulted from a cross-reactionsuspect that some component in the blood collection tubes

17). After the change of transition, a strong

noise peak was detected in the vicinity of the 25(OH)D3

12)

used in Hospital A caused the noise peak.
4

June 2019

Medical Mass Spectrometry Vol. 3 No. 1

Fig. 1. SRM chromatograms for 25(OH)D3 in serum samples collected in two types of blood collection tubes.

The left chromatograms were obtained by LC/MS/MS of a serum sample collected by Chiba University Hospital. The right chromatograms are of a serum sample collected by Hospital A. (a) The transition was set at 383.3 → 105.1 for 25(OH)D3 measurement.
(b) The transition was set at 383.3 → 365.2 for 25(OH)D3 measurement.

As we reported previously , the time intervals between

18)

ponents are extremely complex. Matrix effects means the

venipuncture and serum separation have a signiﬁcant

global impact that the presence of coeluting substances

impact on serum peptide proﬁles. As we demonstrated
more recently, it is essentially important in serum bio-

have on the process of declustering and the ionization ofmarker exploration to consider the inﬂuence of storage tem-matrix effect is mitigated by appropriate sample preparation

19)

clinical mass spectrometry have to consider that a series ofand chromatographic separation. Moreover, insufﬁcient
sample preparation not only shortens the life of the col-operations from sample collection to analysis greatly affectumns for liquid chromatography but also increases thethe test results.

downtime risk of the equipment, because crude samples

20)

analytes within the ion source region . In general, theperature and storage period . Those who are involved inwere injected directly into the equipment. The matrix
effects can be conﬁrmed through post-column infusion ofRole of sample preparation for reduction of matrix
effectAppropriate sample preparation is required to accurately

the target analyte solution into the eluent from the HPLC
21)column via a T-piece . Phospholipids are one of the mostmeasure a target analyte in biological samples whose com-well-known factors of matrix effect, and its elution pattern5

June 2019

Fig. 2.

Medical Mass Spectrometry Vol. 3 No. 1

Matrix effects (most likely due to serum phospholipids) during sample preparation using four methods for derivatized 25(OH)D3 and 3-epi-25(OH)D3.

(a) SRM chromatograms obtained using post-column infusion of a solution containing derivatized 25(OH)D3 and 3-epi-25(OH)D3 at a concentration of 750 and 250 ng/mL, respectively. The infusion rate was 10 μL/min. 20 μL of pooled serum sample wasprepared using each method and injected onto the column using an autosampler. (b) Precursor ion scan at 184 m/z of each preparedsample. (c) SRM chromatogram of lysophosphatidylcholine (C16:0). (d) SRM chromatogram of lysophosphatidylcholine (C18:0).

6

June 2019

Medical Mass Spectrometry Vol. 3 No. 1

can be identiﬁed by precursor ion scan at 184 m/z. We compared four sample preparation methods [protein precipita-

ionization (APCI) and atmospheric pressure photoioniza20)
tion (APPI) . It was recently reported that a new commer-

tion (PP), liquid-liquid extraction (LLE), solid phasecial atmospheric pressure ionization (API) source, UniS-

extraction (SPE) and supported liquid extraction (SLE)]. In
this comparison, LC separation was carried out using a col-

pray

TM

(Waters, USA), slightly reduces the matrix effect23)

compared with ESI, depending on the target analyte .

umn of a reverse phase distribution system, which separates
analytes based on the lipid solubility. Fig. 2 shows the

Stable isotope-labeled internal standards

matrix effects observed using each preparation method forderivatized 25(OH)D3 and 3-epi-25(OH)D3 transition

Internal standards (ISs) are essential to compensate formatrix effects and correct potential variations during sam-olites of vitamin D . Strong ion suppression at the indi-

for selecting ISs and strongly recommends that the IS be a24)
stable isotope-labeled form of the analyte being tested .

(619.50 → 341.2). The HPLC and MS parameters were set
according to our previous report for measuring four metab-

ple preparation steps. The Clinical and Laboratory Standards Institute (CLSI) C62-A lists seven recommendations

10)

cated elution times was detected using PP and SPE whereas
LLE resulted in minimal ion suppression. When the precursor ion scan was performed using the same sample, elution

Deuterium, C, N and O labeled compounds are generally used as ISs, although the elution time of a deuteri-of phospholipids was detected at the elution time whenstrong ion suppression occurred (Fig. 2b). Molecular spe-um-labeled IS is slightly faster than that of the target analyte on reversed-phase separation, requiring carefulcies of phospholipid can be presumed by setting multiple

attention to matrix effects caused by phospholipid and othertransitions with the precursor ion of the phospholipid and a
product ion of 184 m/z. Lyso-PC species containing palmitic

compounds. On the other hand, C, N and O labeled ISs

13

15

18

13

15

18

may be more useful than deuterium labeled IS because they
elute almost adjacent to the target analyte. It is noteworthy
that hydrogen-deuterium exchange may occur, dependingacid (C16:0) or stearic acid (C18:0) were detected with thesame elution time as the species detected by a precursor ion
scan of 184 m/z (Fig. 2c and 2d). The matrix effect was noteffectively removed by SPE, which is most likely because

on the labeled position, resulting in inconsistent IS
25)
responses . Thus, stable isotope-labeled ISs are indispens-

the same reversed phase separation mode was used for SPE

able for clinical mass spectrometry, but deuterium-labeledand LC column. For effective pretreatment, it is important

ISs are not widely available for the majority of potential

to use SPE with a separation mode different from that usedin LC columns, such as ion exchange and mixed-mode.

analytes.

®
HybridSPE -Phospholipid columns (Sigma-Aldrich, Germany) packed with zirconia-coated silica is effective for

Calibrator- and serum-based certiﬁed reference materials

22)

removing phospholipids by SPE . LLE gave the weakest
ion suppression because its separation is based on the polar-

Calibrators serve as references for converting raw data to
the concentration of the target analyte. The use of faulty

ity, unlike the reverse phase distribution mode. Because

calibrators leads to erroneous results. A New York Times

SLE is the same separation mode as LLE, it effectively

article on January 7, 2009 shook the LC/MS/MS society .
A spokesperson from Quest (one of the distinguished pri-26)

removed the matrix effect. However, LLE is not suitable
for large-scale studies because this technique is not auto-vate medical laboratories in the US) admitted that somemated. On the other hand, SPE, SLE and Hybrid®
SPE -Phospholipid can be easily automated. Automatederroneous 25(OH)D results had been reported because ofsample preparation enables consistent sampling procedures.

the problems with calibrators. Although it is generally
believed that inter-laboratory variation in LC/MS/MS mea-

Eventually, complete automation, including data analysis

surements is small compared with immunoassays, that is

and interfacing, will be the key for further acceleration of

not necessarily the case based on the results of the 25(OH)D College of American Pathologists (CAP) survey pre-the clinical application of LC/MS/MS.
Degrees of matrix effects have different degrees of inﬂu-

sented at a workshop held by the FDA in May 2016 . A

ence depend on the ionization mode. Ion suppression isoften observed well in ESI positive mode, but it is hardly

total of 8 immunoassays were included in the 25(OH)DCAP survey, and the method-speciﬁc between-laboratory

seen in ESI negative mode, atmospheric pressure chemical

CVs ranged from 6.4 to 11.6%. As many as 74 laboratories

27)

7June 2019

Medical Mass Spectrometry Vol. 3 No. 1

ments. These calibration problems are not unique to vitamin D measurements and are also important in the measure-reported results obtained using LC-MS/MS and the CV was
11.4%. It is likely that inter-laboratory variation can be
28)

improved if a common calibrator is used . Considering

ment of steroid hormones and other low molecule analytes.
Currently available serum-based CRMs other than vitaminthat the results of 25(OH)D submitted to the International
Quality Assessment Scheme (DEQAS) have shown poor

D metabolites are limited and include SRM971 for cortisol,
testosterone, progesterone, total T3 and total T4; BCR576‒

interlaboratory agreement, Carter and Jones tried to answer
whether the use of a common standard could reduce the dis-

578 for 17beta-estradiol; ERMDA192‒193 for cortisol;29)

cordance . They demonstrated that use of a commercially

ERMDA347 and BCR348R for progesterone; NMIJ CRM
6401-b for cortisol; and NMIJ CRM 6402-a for aldosterone.

available common standard (Chromsystems Inc., Germany)
reduced the mean inter-laboratory precision (CV) for

More serum-based CRMs are needed to accelerate theimplementation of MS-based assays into clinical laborato-

25(OH)D3 from 16.7% (in-house standard) to 8.5% (common standard). On the other hand, it was reported that cali-ries. CLSI released a standardized approach for LC-MS

bration alone is not the cause of variation in LC/MS/MS

assay development and veriﬁcation in its 2014 guidance

30)

testosterone measurement . Although it is desirable that

document, mainly for low molecule compounds. Similar

the matrix for the calibrator be the same as that for realguidance documents for proteins and peptides are required.

samples, this is not possible when the target analytes areendogenous compounds. Successful quantitation of endoge-

Data analysis and instrument control software

nous analytes in biological samples by LC/MS/MS without

The use of updated LC/MS/MS platforms, and deﬁning

a true blank is challenging. One approach is to pretreat sera
samples to remove as much the particular endogenous ana-

the peak detection method, allow automatic peak detection
and quantiﬁcation. Several different peak detection algo-lyte as possible, which is not satisfactory under most circumstances. An alternative approach is to use a deuteri-rithms are adopted for available MS instruments, but

um-labeled compound as a surrogate analyte for generating

whether the results obtained automatically agree well withregarding the validity thereof, it is necessary to check31)

calibration curves .
BSA solutions (4‒7%) are often used as a matrix substi-

those obtained manually by LC/MS experts. LC/MS

tute, but the possibility that BSA solutions contain any

experts check not only peak area ratio of analyte and IS but
also analyte area of all QC samples and IS area of all speci-

binding proteins to the target analyte should be noted.

mens independently. Few software programs automatically

Large deviations in 25(OH)D3 values might be caused by29)
vitamin D-binding protein contaminants in BSA . Further-plot the area value of the analyte and IS, and thus this must

more, we have detected signiﬁcant amounts of 25(OH)D3
in commercially available non-recombinant BSA (data not

Making LC/MS/MS platforms more applicable to routine

be done manually at present.

published). Commercially available recombinant humanclinical laboratory analyses will require total automation,
including automated sample preparation, sophisticated soft-

serum albumin (HSA) does not contain detectable amounts

ware for data review, and interfacing with laboratory infor-of 25(OH)D3 and can be used as a matrix. It is important tomation systems. Mass spectrometry platform manufacturers
conduct their standard performances tests with easy-to-han-note not only the type of matrix used for the calibrator but
also the purity of the standard materials per se. Purity index
may differ depending on the determination method (fordle analytes to ensure the function of the instrument but do
not go beyond that. Indeed, a considerable in-house capabil-example, HPLC and quantitative NMR).

ity for troubleshooting and maintenance of the MS instru-

Clearly, it is impossible to obtain accurate calibration
curves using impure materials, thus highlighting the impor-ment is currently required. As pointed out by Vogeser andSeger, software-based troubleshooting and maintenance

32)
tance of certiﬁed reference materials (CRMs) . Solvent-based standard materials (SRM 2972a) and reference

instruction tools would be effective but are not currently

sera (SRM 972a) are currently available. The National
Institute of Standards and Technology (NIST) supplies ref-At present, MS assays are mostly laboratory-developed,
labor-intensive, and require considerable expertise inerence sera with values assigned by LC-MS/MS, and theseare helpful in assuring the accuracy of 25(OH)D measure-method development and validation. As LC/MS/MS procedures become more automated and more MS-related in33)

available .

8June 2019

Medical Mass Spectrometry Vol. 3 No. 1

vitro diagnostics become commercially available, the application of LC/MS/MS to laboratory medicine will be signiﬁ-

98: 3971‒3973, 2013.
9) Ihara H, Kiuchi S, Ishige T, Nishimura M, Matsushita K,
et al: Surveillance evaluation of the standardization of as-

cantly accelerated.

say values for serum total 25-hydroxyvitamin D concen-

Acknowledgments

tration in Japan. Ann Clin Biochem 55: 647‒656, 2018.

This research was partially supported by the Ministry of
Education, Culture, Science, Sports and Science and Tech-

10) Satoh M, Ishige T, Ogawa S, Nishimura M, Matsushita K,

nology (MEXT) by a Grant-in-Aid for Scientiﬁc Research(C), 2018‒2021, 18K07462 (M. Satoh).

measurement of four vitamin D metabolites in serum by

et al: Development and validation of the simultaneous
LC-MS/MS for clinical laboratory applications. Anal Bioanal Chem 408: 7617‒7627, 2016.

Conﬂict of Interest11) Nishikawa T, Omura M, Kawaguchi M, Takatsu A, SatohAll authors declare that they have no conﬂict of interest.

F, et al: Calibration and evaluation of routine methods byserum certiﬁed reference material for aldosterone mea-

References

surement in blood. Endocr J 63: 1065‒1080, 2016.

1) Meier F, Geyer PE, Virreira Winter S, Cox J, et al: Box-

12) Satoh M, Miyabayashi Y, Seimiya M, Kazami T, Ishige T,

Car acquisition method enables single-shot proteomics at

et al: Development and validation of the simultaneous

a depth of 10,000 proteins in 100 minutes. Nat Methods

measurement of estrone and 17-b-estradiol in serum by

15: 440‒448, 2018.

LC-MS/MS for clinical laboratory applications. Medical

2) Jarmusch AK, Pirro V, Baird Z, Hattab EM, Cohen-Gadol

Mass Spectrometry 3: 1‒11, 2019.

AA, et al: Lipid and metabolite proﬁles of human brain

13) Vogeser M, Seger C: Pitfalls associated with the use oftumors by desorption electrospray ionization-MS. Proc

liquid chromatography-tandem mass spectrometry in theNatl Acad Sci 113: 1486, 2016.

clinical laboratory. Clin Chem 56: 1234‒1244, 2010.

3) Yoshimura K, Chen LC, Johno H, Nakajima M, Hiraoka

14) Ishige T, Satoh M, Ogawa S, Nishimura M, Matsushita K,

K, et al: Development of non-proximate probe electro-

et al: Improved sensitivity of serum/plasma 1alpha,25-di-

spray ionization for real-time analysis of living animal.

hydroxyvitamin D quantiﬁcation by DAPTAD derivatiza-

Mass Spectrom 3: S0048‒S0048, 2015.

tion. Clin Chim Acta 473: 173‒179, 2017.

4) St John ER, Balog J, McKenzie JS, Rossi M, Covington

15) Anderson NL, Anderson NG, Haines LR, Hardie DB,

A, et al: Rapid evaporative ionisation mass spectrometry

Olafson RW, et al: Mass spectrometric quantitation ofof electrosurgical vapours for the identiﬁcation of breast

peptides and proteins using stable isotope standards andpathology: Towards an intelligent knife for breast cancer

capture by anti-peptide antibodies (SISCAPA). J Pro-surgery. Breast Cancer Res 19: 59, 2017.

teome Res 3: 235‒244, 2004.

5) Brodin P: The biology of the cell-insights from mass cy-16) Wang C, Shiraishi S, Leung A, Baravarian S, Hull L, ettometry. FEBS J: https://doi.org/10.1111/febs.14693, 2018.

al: Validation of a testosterone and dihydrotestosterone

6) Nomura F: Proteome-based bacterial identiﬁcation using

liquid chromatography tandem mass spectrometry assay:

matrix-assisted laser desorption ionization-time of ﬂight

Interference and comparison with established methods.

mass spectrometry (MALDI-TOF MS): A revolutionary

Steroids 73: 1345‒1352, 2008.

shift in clinical diagnostic microbiology. Biochim Biophys17) Mochizuki A, Kodera Y, Saito T, Satoh M, Sogawa K, etActa 1854: 528‒537, 2015.

al: Preanalytical evaluation of serum 25-hydroxyvitamin
D3 and 25-hydroxyvitamin D2 measurements using LC-

7) Seng P, Drancourt M, Gouriet F, La Scola B, Fournier PE,
et al: Ongoing revolution in bacteriology: Routine identi-

MS/MS. Clin Chim Acta 420: 114‒120, 2013.

ﬁcation of bacteria by matrix-assisted laser desorption18) Umemura H, Nezu M, Kodera Y, Satoh M, Kimura A, etionization time-of-ﬂight mass spectrometry. Clin Infectal: Effects of the time intervals between venipuncture andDis 49: 543‒551, 2009.

serum preparation for serum peptidome analysis by ma-8) Handelsman DJ, Wartofsky L: Requirement for mass

trix-assisted laser desorption/ionization time-of-ﬂight

spectrometry sex steroid assays in the Journal of Clinicalmass spectrometry. Clin Chim Acta 406: 179‒180, 2009.

Endocrinology and Metabolism. J Clin Endocrinol Metab

19) Tsuchida S, Satoh M, Umemura H, Sogawa K, Takiwaki
9

June 2019

Medical Mass Spectrometry Vol. 3 No. 1

M, et al: Assessment by matrix-assisted laser desorption/

26) Pollack A: Quest acknowledges errors in vitamin D tests.

ionization time-of-ﬂight mass spectrometry of the effects

New York Times, 2009.

of preanalytical variables on serum peptidome proﬁles27) Hoofnagle A: Using Mass Spectrometry to Help Patients.

following long-term sample storage. Proteomics Clin

Public Workshop̶Mass Spectrometry in the Clinic: Reg-

Appl 12: 1700047, 2018.

ulatory Considerations Surrounding Validation of Liquid20) Taylor PJ: Matrix effects: The Achilles heel of quantita-

Chromatography-Mass Spectrometry Based Devices.

tive high-performance liquid chromatography-electro-

http://waybackarchive - itorg/7993/20171115183116/

spray-tandem mass spectrometry. Clin Biochem 38: 328‒

https://wwwfdagov/downloads/MedicalDevices/NewsEv

334, 2005.

ents/WorkshopsConferences/UCM499238pdf, 2016.

21) Annesley TM: Ion Suppression in Mass Spectrometry.

28) Yates AM, Bowron A, Calton L, Heynes J, Field H, et al:

Clin Chem 49: 1041‒1044, 2003.

Interlaboratory variation in 25-hydroxyvitamin D2 and

22) Pucci V, Di Palma S, Alﬁeri A, Bonelli F, Monteagudo E:

25-hydroxyvitamin D3 is signiﬁcantly improved if com-

A novel strategy for reducing phospholipids-based matrix

mon calibration material is used. Clin Chem 54: 2082‒

effect in LC-ESI-MS bioanalysis by means of HybridSPE.

2084, 2008.

J Pharm Biomed Anal 50: 867‒871, 2009.

29) Carter GD, Jones JC: Use of a common standard im-23) Lubin A, De Vries R, Cabooter D, Augustijns P, Cuyckens

proves the performance of liquid chromatography-tandem

F: An atmospheric pressure ionization source using a highmass spectrometry methods for serum 25-hydroxyvita-

voltage target compared to electrospray ionization for the

min-D. Ann Clin biochem 46: 79‒81, 2009.

LC/MS analysis of pharmaceutical compounds. J Pharm

30) Owen LJ, MacDonald PR, Keevil BG: Is calibration the

Biomed Anal 142: 225‒231, 2017.

cause of variation in liquid chromatography tandem mass

24) Institute. CaLS: C62-A liquid chromatography-mass spec-

spectrometry testosterone measurement? Ann Clin Biochem 50: 368‒370, 2013.

trometry methods; Approved Guideline. https://clsiorg/
standards/products/clinical - chemistry - and - toxicology/31) Li W, Cohen LH: Quantitation of endogenous analytes in
bioﬂuid without a true blank matrix. Anal Chem 75:

documents/c62/ (Accessed May 2016), 2014.
25) Chavez-Eng CM, Constanzer ML, Matuszewski BK:

5854‒5859, 2003.

High-performance liquid chromatographic-tandem mass

32) Carter GD: 25-hydroxyvitamin D assays: The quest forspectrometric evaluation and determination of stable iso-accuracy. Clin Chem 55: 1300‒1302, 2009.

tope labeled analogs of rofecoxib in human plasma sam-33) Vogeser M, Seger C: A decade of HPLC-MS/MS in theples from oral bioavailability studies. J Chromatogr B An-

routine clinical laboratory̶Goals for further develop-

alyt Technol Biomed Life Sci 767: 117‒129, 2002.

ments. Clin Biochem 41: 649‒662, 2008.

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