What is novel about certified reference materials?
Analytical and Bioanalytical Chemistry
What is novel about certified reference materials?
Stephen A. Wise 0
Stephen A. Wise 0
0 Office of Dietary Supplements, National Institutes of Health (NIH-ODS) , Bethesda, MD , USA
the years, I have had numerous discussions with colleagues at
NIST and elsewhere (including ABC editors) concerning the
merits of publishing CRM papers and specifically what are the
criteria for analytical novelty regarding CRMs. This Editorial
will address the question of analytical novelty associated with
CRM papers by highlighting some recent examples published
in ABC and other analytical chemistry journals.
Analytical and Bioanalytical Chemistry (ABC) has had a
long tradition of publishing papers related to the development
of reference materials. Beginning with its predecessors,
Fresenius’ Zeitschrift für Analytische Chemie and continuing
with Fresenius’ Journal of Analytical Chemistry, ABC has
published topical collections of papers related to reference
materials, with the most recent collection assembled in 2015
]. One important criterion for papers published in ABC, as
well as in other analytical journals, is Banalytical novelty^,
which is typically directed to the methods reported in the
paper. For papers describing CRM development, the scope
of analytical novelty extends beyond just the analytical
methods used to characterize the CRMs to include also: (1)
new or unique CRMs, e.g., new matrices or analytes, and (2)
novel approaches for preparing reference materials. Finally,
many CRM papers describe the complete CRM development
process including preparation, homogeneity and stability
assessment, and assignment of certified values. In my opinion
(which may be biased because of my CRM career), the
development process for CRMs, because of its rigor and high
metrological approach, represents analytical novelty.
Why do NMIs publish papers describing CRMs? CRM
producers are required to publish a Bcertification report^ describing
the preparation, homogeneity testing, stability assessment,
analytical measurements, and value assignment approach for each
CRM. Although this requirement is satisfied in different ways
and to different degrees by the various CRM producers, one
way to fulfill the requirement is to publish a peer-reviewed
paper describing the CRM development process. The rigorous
details of the CRM development may be overwhelming for
most analytical journals. However, supplemental material now
provides an option to include many of these details. Another
reason to publish CRM papers is to promote their availability
and use. Why should ABC’s readers be interested in papers
describing CRMs? Analytical chemists should be aware of
what CRMs are available to support their analytical
measurements, and they should understand and appreciate the rigor and
exactness involved in their production.
Novel matrix CRMs
Many of the recent CRM papers describe new (first time) and
unique materials, and thereby qualify as novel even if the
analytical methods used to characterize the CRMs may be
wellestablished. NIST has a long history of producing synthetic
greenhouse gas mixture SRMs (CO2, CH4, and N2O) based
on gravimetric preparation. Recently, Rhoderick et al. [
reported the first CRMs for whole natural air with values
assigned for greenhouse gases, denoted as SRM 1720
Northern Hemisphere Air [
] and SRM 1721 Southern
Oceanic Air . These unique SRMs consist of batches of 30
cylinders of pristine ambient air collected at two remote
locations: (1) Niwot Ridge (CO, USA) for SRM 1720 and Baring
Head, New Zealand for SRM 1721. Rhoderick et al. [
described the process of assigning certified values to each
individual cylinder based on calibrating the instrumentation using a
suite of gravimetrically-prepared NIST primary reference gas
mixtures. The nominal values for the three gases were nearly
identical for CO2 (388 μmol/mol) and N2O (323 μmol/mol) in
both SRMs, and the value for CH4 in SRM 1720 is 5.6% higher
(1860 μmol/mol). The relative uncertainties associated with the
certified values were <0.06%, representing the smallest
uncertainties on any NIST gas SRM.
Another first-time CRM matrix, cigarette tobacco filler,
was introduced by Sander et al. [
]. SRM 3222 Cigarette
Tobacco Filler has certified values assigned for nicotine, two
tobacco-specific nitrosamines [N-nitrosonornicotine (NNN)
and 4-(methylnitrosomino)-1-(3-pridyl)-1-butanone (NNK)].
The certified values for nicotine, NNN, and NNK were
assigned by combining the results from several different
sample preparation approaches followed by isotope dilution (ID)
liquid chromatography – tandem mass spectrometry (LC-MS/
Not all CRM papers describe new or first-time matrices or
analytes to qualify as novel. For example, Phinney et al. [
do not describe a new CRM but instead satisfy the novelty
requirement by offering a significantly Bimproved^ material.
In 2012 Phinney et al. [
] reported the first serum-based
CRM with values assigned for three vitamin D metabolites,
i.e., 25-hydroxyvitamin D2 [25(OH)D2], 25-hydroxyvitamin
D3 [25(OH)D3], and 3-epi-25-hydroxvitamin D3
[3-epi25(OH)D3]. SRM 972 Vitamin D in Human Serum was a
huge success and the inventory was rapidly exhausted. In
2017 Phinney et al. [
] introduced a new, improved SRM
972a Vitamin D Metabolites in Human Serum to replace
SRM 972. What were the significant improvements to satisfy
the analytical novelty criteria? The original material was
designed to provide typical analytical challenges encountered in
the determination of total 25-hydroxyvitamin D [i.e., the sum
of 25(OH)D2 and 25(OH)D3] in clinical patient samples.
SRM 972 consisted of four levels of vitamin D metabolites
representing normal and low levels of 25(OH)D3, high levels
of 25(OH)D2, and high levels of 3-epi-25(OH)D3, which
must be chromatographically separated from 25(OH)D3 for
accurate measurement of total 25(OH)D. To achieve target
levels, the normal level was diluted with horse serum, which
contains no 25(OH)D, to provide a low level, and the
remaining two levels were fortified with either 25(OH)D2 or
3-epi25(OH)D3. Even though SRM 972 was widely used, a major
criticism was that the use of modified serum and/or the
presence of non-endogenous metabolites in three of the four
levels produced inaccurate results using some assays,
In SRM 972a only one of four levels contained
nonendogenous metabolites, i.e., only addition of 3-epi-25(OH)D3.
The low level and normal levels of 25(OH)D3 were from donor
pools that were screened to provide target levels, and the high
level of 25(OH)D2 was achieved through donors supplemented
with vitamin D2. Another significant improvement for SRM
972a was the use of only LC-MS/MS methods that separated
the 25(OH)D3 and 3-epi-25(OH)D3 isomers to assign the
certified values. Finally, the paper also contained results addressing
the commutability of SRM 972a, i.e., does it behave the same as
clinical patient samples, which is an important aspect for clinical
CRMs. Tai et al. [
] described a related new SRM with a high
level of 25(OH)D3 (25% higher than in SRM 972a), SRM 2973
Vitamin D Metabolites in Serum (High Level). The paper’s
novelty was based on the first certified values in both SRM 2973 and
SRM 972a for another important metabolite,
24R,25dihydroxyvitamin D3 [24,25(OH)2D3], using an ID LC-MS/
MS reference measurement procedure.
Novel CRM production
NIST and JRC have invested considerable effort in developing
CRMs to support measurements of trace elements and organic
pollutants in fine particulate matter (PM). Two of the earliest
and most widely-used NIST environmental-matrix SRMs are
based on PM, i.e., SRM 1648 Urban Particulate Matter and
SRM 1649 Urban Dust, which were issued in 1978 and 1982,
respectively. Both SRMs were prepared from large quantities
of total suspended particulate (TSP) matter (< 63 μm)
collected over 18-month periods. Because of the significant
quantities collected, these two SRMs are still available today after
nearly 40 years. However, because of the need for
contemporary PM and the potential hazards and regulations for PM with
aerodynamic diameter < 10 μm (PM10) and <2.5 μm (PM2.5),
Schantz et al. [
] reported the development of the first natural
fine PM CRMs, i.e., SRM 2786 Fine Particulate Matter
(< 4 μm) and SRM 2787 Fine Particulate Matter (< 10 μm),
for determination of polycyclic aromatic hydrocarbons
(PAHs), nitro-substituted PAHs, brominated diphenyl ether
congeners, and trace elements. Schantz et al. [
the multiple analytical methods used (e.g., different extraction
techniques and gas chromatography/mass spectrometry (GC/
MS) on columns of different selectivity for the organic
contaminants). Obtaining a sufficient quantity of fine PM to
produce a CRM is a significant challenge. After attempts to
collect ambient PM2.5, NIST ultimately resorted to resuspending
TSP using a particle suspension unit and ultra-high-volume
sampler to collect the size-fractionated PM.
JRC has also devoted substantial time and thought to
obtaining the required quantity of PM2.5 to produce a CRM.
Initial efforts at JRC produced two CRMs for PM10-like
materials, ERM-CZ100 [
] and ERM-CZ120 , which are
certified for PAHs and toxic heavy metals, respectively. Both
materials were prepared by jet-milling dust collected from a
road tunnel, hence the designation as BPM10-like^ materials.
As the next step in the quest for producing PM2.5 CRMs
Emteborg and coworkers [
] describe a novel process for
preparing PM2.5-like material intended for the determination
of anions (SO42−, NO3−, Cl−,) and cations (Ca+, K+, NH4+,
Ca2+, and Mg2+). Starting with the TSP used for ERM-CZ100
and ERM CZ-120, a PM10-like material was produced by
jetmilling and then suspended in water. After sedimentation of
the larger particles, 90% by volume was siphoned off, and the
remaining solution was spiked with the anions and cations
prior to drop-wise shock freezing in liquid nitrogen. The
resulting ice kernels were then freeze-dried. Using this
process, it was possible to produce 500 g of PM2.5-like material.
The authors envision using this process to produce future
PM2.5-like materials for PAHs and trace elements.
Obviously, a CRM paper can be considered as novel based on
the methods developed and used to certify the CRM. Phillips
et al. [
] described the use of two novel independent methods
for the determination of isoflavones in four different
soybased dietary supplement SRMs. Different sample preparation
approaches were optimized followed by analysis using LC
with absorbance detection and ID LC-MS for the
determination of six isoflavones in soy flour, soy protein isolate, soy
protein concentrate, and soy-containing oral solid dosage
form. Phillips et al. [
] compared the results from the two
analytical methods, which also contributes to the analytical
novelty, and described the combination of results to obtain
the certified values.
The complete CRM development process
My final classification is papers describing the complete CRM
development process, i.e., production, homogeneity and
stability assessment, and value assignment approach. Several
excellent examples of these Bcomplete process^ papers are
highlighted below. Since the early 1990s, NRCC Halifax,
the premier NMI for marine biotoxin research, has produced
both calibration solution and shellfish tissue-matrix CRMs for
a wide variety of biotoxins. Recently, two companion papers
from NRCC reviewed developments in the production of
CRMs for diarrhetic shellfish poisoning toxins (DSPs).
Beach et al.  summarized the current approach for
production of calibration solution CRMs for DSPs, i.e., isolation and
purification of sufficient quantities of DSPs from algal
cultures and characterization and purity assessment using
quantitative nuclear magnetic resonance (NMR). After stability
and homogeneity assessments, final quantification was
performed by LC-MS/MS. In the companion paper, McCarron
et al.  described the development of a new mussel tissue
CRM with naturally-incurred levels of DSPs.
A series of papers describing development of a new
freezedried mussel tissue material (CRM-FDMT1) for multiple
phycotoxins was published by McCarron and coworkers
[16–19]. The papers described the various aspects of CRM
development including design and preparation ; LC-MS/
MS, extraction, and quantification approaches ;
homogeneity and stability  and certification . Prior to the
development of CRM-FDMT1, mussel tissue CRMs were
available only for single toxin groups. CRM-FDMT1 was prepared
as a mixture of five batches of mussels from different locations
and contaminated with six different toxin groups, i.e., domoic
acid, DSPs, azapiracids, pectenotoxins, yessotoxinsm, and
spirolides. For the certification measurements, LC-MS/MS
methods, which were optimized for each toxin group, were
employed following exhaustive extraction procedures . To
compensate for LC-MS/MS matrix effects, a variety of
quantification approaches were used including standard addition,
dilution, or matrix-matched calibration. CRM-FDMT1 is the
first shellfish-tissue matrix CRM with values assigned for 10
toxins from multiple groups, and the first CRM with values
assigned for yessotoxins, pectenotoxins, and spirolides.
Papers describing development of CRMs for the
determination of trace elements may be criticized as having limited
analytical novelty, since the analytical techniques used are
often well-established or even routine methods. A recent
paper by Yu et al.  describing a kelp powder SRM is, in my
opinion, a excellent model for a trace element CRM paper.
SRM 3232 Kelp Powder (Thallus laminariae) was developed
to support measurements for compliance with food and
dietary supplement regulations. Kelp was selected because it is a
rich source of dietary iodine and vitamin K1, and it contains
high levels of total arsenic, including both toxic inorganic
species and less toxic organic species. Yu et al.  explained
the need for the kelp material and compared the elemental
content of the kelp powder SRM to similar seaweed-matrix
CRMs from other NMIs. Using multiple analytical
techniques, certified values for 15 nutritional and toxic elements
were assigned. Most importantly regarding analytical novelty,
values are assigned for arsenic species, including arsenic acid,
dimethylarsinic acid, and three arsenosugars using a novel
combination of LC with instrumental neutron activation
analysis (INAA) and LC with inductively coupled plasma/mass
spectrometry (ICP/MS). SRM 3232 is the first
seaweedmatrix CRM with values assigned for iodine, arsenic species
(including arsenosugars), and cis- and trans-isomers of
vitamin K1, which were determined using ID LC-MS/MS.
Another example of the rigor of the CRM development
process is reported by Merrick and coworkers  from NMIA
describing the production of a coastal seawater CRM (NMIA
MX014) with certified values for 12 elements, which were added
to the natural sea water sample to provide levels relevant to the
Australian environmental regulations. Values were assigned
using different sample preparation, ID and standard addition
quantification approaches, and various ICP/MS techniques.
NIST and JRC have the longest history and broadest scope
of CRMs for chemical composition, and they have worldwide
distribution and CRM brand recognition. Some NMIs choose
to focus on only a limited chemical measurement area (e.g.,
the case of marine biotoxins at NRCC Halifax described
above). Other NMIs with more recently-established chemical
metrology programs (e.g., NMIJ, KRISS, NIMC) are making
significant contributions to CRM production; however, in
most cases, these CRMs are intended for use only in the
country of origin. For example, recent papers from NIMC report
the production of CRMs for the determination of clinical
markers in human serum. L. Feng et al.  and Y. Lui et al.
 described the certification of human serum-based CRMs
for the determination of electrolytes and total homocysteine,
respectively, using higher-order methods, which were
validated through international comparisons with other NMIs and
through comparisons with existing CRMs. The CRMs
reported in these papers are not new or novel materials (other NMIs
have produced similar materials); however, these CRMs are
significant in that they demonstrate the high level of chemical
metrology practiced at NIM China.
Finally, it is noteworthy that many of the papers highlighted in
this Editorial have an extensive list of coauthors; most papers
have more than six coauthors and two papers have 15 coauthors.
The development of a natural-matrix CRM is a massive
undertaking that generally involves multiple analytical techniques,
numerous analysts, statisticians, and often collaborators with unique
capabilities from other institutions.
I hope that this Editorial has stimulated your interest in
papers describing CRMs and has emphasized the various
aspects of novelty associated with these valuable analytical
tools. I will continue to encourage my colleagues at NMIs
worldwide to submit high-quality papers to ABC describing
their CRM development activities, and I hope that ABC
readers will continue to find these papers to be beneficial.
1. International Standards Organization (ISO) ( 2016 ) General requirements for competence of reference material producers . ISO Guide 17034.
2. Wise SA , Emons H . Reference materials for chemical analysis . Anal Bioanal Chem . 2015 ; 407 ( 11 ): 2941 - 3 . https://doi.org/10. 1007/s00216-015-8500-1.
3. Rhoderick GC , Kitzis DR , Kelley ME , Miller WR , Hall BD , Dlugokencky EJ , et al. Development of a northern continental air standard reference material . Anal Chem . 2016 ; 88 ( 6 ): 3376 - 85 . https://doi.org/10.1021/acs.analchem.6b00123.
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5. Sander LC , Pritchett JS , Daniels YC , Wood LJ , Lang BE , Wise SA , et al. Development of a cigarette tobacco filler standard reference material . Anal Chem . 2017 ; 89 ( 19 ): 10461 - 7 . https://doi.org/10. 1021/acs.analchem.7b02550.
6. Phinney KW , Tai SSC , Bedner M , Camara JE , Chia RRC , Sander LC , et al. Development of an improved standard reference material for vitamin D metabolites in human serum . Anal Chem . 2017 ; 89 ( 9 ): 4907 - 13 . https://doi.org/10.1021/acs.analchem.6b05168.
7. Phinney KW , Bedner M , Tai SSC , Vamathevan VV , Sander LC , Sharpless KE , et al. Development and certification of a standard reference material for vitamin D metabolites in human serum . Anal Chem . 2012 ; 84 ( 2 ): 956 - 62 . https://doi.org/10.1021/ac202047n.
8. Tai SSC , Nelson MA , Bedner M , Lang BE , Phinney KW , Sander LC , et al. Development of standard reference material (SRM) 2973 vitamin D metabolites in frozen human serum (high level) . J AOAC Int . 2017 ; 100 ( 5 ): 1294 - 303 . https://doi. org/10.5740/jaoacint.17- 0182 .
9. Schantz MM , Cleveland D , Heckert NA , Kucklick JR , Leigh SD , Long SE , et al. Development of two fine particulate matter standard reference materials (< 4 μm and < 10 μm) for the determination of organic and inorganic constituents . Anal Bioanal Chem . 2016 ; 408 ( 16 ): 4257 - 66 . https://doi.org/10. 1007/s00216-016-9519-7.
10. Certification Report ERM-CZ100 ( 2010 ) The certification of the mass fraction of selelcted polycyclic aromatic hydrocarbons (PAHs) in fine dust (PM10-like matrix) . ISBN 978-52-79-17528-2.
11. Certification Report ERM-CZ120 ( 2010 ) The certification of the mass fractions of arsenic, cadmium, nickel, and lead in fine dust (PM10-like matrix) . ISBN 978-52-79-17527-5.
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McCarron P , Giddings SD , Quilliam MA . A mussel tissue certified reference material for multiple phycotoxins. Part 2: liquid chromatography-mass spectrometry, sample extraction and quantitation procedures . Anal Bioanal Chem . 2011 ; 400 ( 3 ): 835 - 46 .
McCarron P , Wright E , Emteborg H , Quilliam MA . A mussel tissue certified reference material for multiple phycotoxins. Part 4: certification . Anal Bioanal Chem . 2017 ; 409 ( 1 ): 95 - 106 . https://doi.org/ 10.1007/s00216-016-0004-0.
Yu LL , Browning JF , Burdette CQ , Caceres KD , Davis WC , Ellisor MB , et al. Development of a kelp powder (Thallus laminariae) standard reference material . Anal Bioanal Chem . 2018 ; 410 ( 4 ): 1265 - 78 .
Merrick JP , Saxby DL , White I , Antin L , Murby EJ . Production of trace elements in coastal sea water certified reference material NMIA MX014 . Anal Bioanal Chem . 2016 ; 408 ( 16 ): 4413 - 24 .
Feng L , Wang J , Cui Y , Shi N , Li H , Li H . Development of certified reference materials for electroytes in human serum (GBW09124- 09126) . Anal Bioanal Chem . 2017 ; 409 ( 13 ): 3483 - 93 . https://doi.
Lui Y , Song D , Xu B , Li H , Dai X , Chen B . Development of a matrix-based candidate reference material of total homocysteine in human serum . Anal Bioanal Chem . 2017 ; 409 ( 13 ): 3329 - 35 . https:// doi.org/10.1007/s00216-017-0272-3. Stephen A. Wise is a Scientist Emeritus at the National Institute of Standards and Technology (NIST) in Gaithersburg, Maryland. During his 40-year career at NIST, he was primarily involved in the development of Standard Reference Materials (SRMs) for trace organic constituents in environmental, clinical, food, and dietary supplement m a t r i c e s . H e i s c u r r e n t l y a Scientific Consultant in the Office of Dietary Supplements at the National Institutes of Health (NIHODS) where he provides support for the Analytical Methods and Reference Materials Program. For his contributions to the development of SRMs, he received the 2006 Harvey W . Wiley Award from AOAC International, the 2014 Reference Material Achievement Award from the Technical Division on Reference Materials of AOAC International, and the 2015 Hillebrand Prize from the Chemical Society of Washington. He is one of the founding editors of Analytical and Bioanalytical Chemistry .