The role of the CCQM OAWG in providing SI traceable calibrators for organic chemical measurements

Abstract

Metrological traceability for organic chemical measurements is a documented unbroken chain of calibrations with stated uncertainties that ideally link the measurement result for a sample to a primary calibrator in appropriate SI units (e.g., mass fraction). A comprehensive chemical purity determination of the organic calibrator is required to ensure a true assessment of this result. We explore the evolution of chemical purity capabilities across metrology institute members of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology’s Organic Analysis Working Group (OAWG). The OAWG work program has promoted the development of robust measurement capabilities, using indirect “mass balance” determinations via rigorous assessment of impurities and direct determination using quantitative nuclear magnetic resonance spectroscopy methods. A combination of mass balance and qNMR has been shown to provide a best practice approach. Awareness of the importance of the traceability of organic calibrators continues to grow across stakeholder groups, particularly in key areas such as clinical chemistry where activities related to the Joint Committee for Traceability in Laboratory Medicine have raised the profile of traceable calibrators.

Introduction

Metrological traceability for organic chemical measurements requires a documented unbroken chain of calibrations with specified uncertainties that ideally link the measurement result for a sample and the measurement procedure to a primary calibrator characterized by an assigned value stated in International System of Units (SI) [1, 2]. For organic measurements, these are typically the derived unit of mass fraction (e.g., mg/g). It is the purity assessment methodology that links the primary calibrator’s assigned value to the SI. The development of appropriate methodologies represents a core competency for all National Metrology Institutes and Designated Institutes (NMI/DI) to provide metrologically sound organic measurements as part of their measurement services.

The Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM) Organic Analysis Working Group (OAWG) upholds the critical evaluation and benchmarking of NMI/DI capabilities for the execution of higher-order measurement procedures for well-defined organic molecular entities [3]. Our domain encompasses chemical measurements for a range of organic measurands (such as vitamins, drugs, hormones, preservatives and contaminants), from neat and solution-based calibrator standards to mass fraction determinations in an array of complex industrial, environmental, bio-fluid and food-based matrices [4].

Irrespective of the analytical techniques used or the nature of the sample, the responsibility of the OAWG is to ensure that measurement results are metrologically traceable to a primary calibrator that can be described in clear units of SI. This must be combined with effective validation of the broader measurement procedure in order to ensure accurate results. The core activities of the OAWG thus cover the chemical purity determination of organic primary calibrators combined with the rigorous assessment of possible biases in methods used for the analysis of more complex matrices.

Chemical purity evaluation within the CCQM is not exclusive to the OAWG, as drivers for SI traceability exist for gas, inorganic, electrochemical, protein and nucleic acid measurement communities that face similar metrological tasks. The metrological hierarchy for many of these measurements have at their apex relevant calibrator materials and require metrologically sound assessments of purity of the measurand of interest [5].

Key stakeholders and international requirements for metrological traceability of pure organic calibrators

A joint intergovernmental organization declaration on metrological traceability was signed in 2011 through cooperation between the International Bureau of Weights and Measures (BIPM), the International Organization of Legal Metrology (OIML), the International Laboratory Accreditation Cooperation (ILAC) and the International Standards Organization (ISO) [6]. This declaration states that “metrological traceability embodies the concepts of measurement uncertainty and calibrations against a hierarchy of reference standards.” Pure organic calibrators are affixed to the top of this hierarchy and are essential to effective implementation of metrologically sound traceability chains of organic measurands.

The ILAC Policy on the Traceability of Measurement Results, ILAC P10:01/2013 [7] describes their strategy regarding the establishment of metrological traceability as required by ISO/IEC 17025 General requirements for the competence of testing and calibration laboratories and ISO 15189 Medical laboratories—Requirements for quality and competence. These ISO standards cover the essential elements to ensure quality and demonstrated competence for calibration, general testing and medical testing laboratories. ILAC P10 defines requirements for traceability provided through certified reference materials (CRMs) and describes as its first pathway traceability being provided by the values assigned to CRMs produced by NMI/DIs and included in the BIPM Key Comparison Database [KCDB] or produced by an accredited RMP under its accredited scope of accreditation. Thus, one outcome of the work carried out by the OAWG is to underpin CRM entries captured in the KCDB. This is essential in ensuring the consistency and comparability of the traceability chain for accredited testing laboratories relying on CRMs from different reference material producers to provide services in organic analysis internationally across a wide range of fields.

The Joint Committee for Traceability in Laboratory Medicine (JCTLM) was established in 2002 through a declaration of cooperation between the BIPM, ILAC and the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC). The ILAC P10 policy document also states that the values assigned to CRMs covered by entries in the JCTLM database are considered to have established traceability. The JCTLM was established in response to the implementation of the European Community (EC) Directive 98/79/EC on in vitro medical devices. This EC Directive increased the focus on traceability requirements within the laboratory medicine community by requiring that “the traceability of values assigned to calibrators and/or control materials must be assured through available reference measurement procedures and/or available reference materials of a higher order” and its evolution into an EC Regulation has continued this trend.

The JCTLM maintains a database of “higher-order reference materials” for the laboratory medicine community. Of the 78 high purity materials listed in the database, 64 are within the remit of the OAWG and all of these are primary calibrators produced by NMI/DIs.

The IFCC promotes standardization activities within the clinical community and has several working groups, such as the Vitamin D Standardization Program WG, focused on these activities [8]. A critical initial criterion in the international standardization of clinical analytes is the availability of primary calibrators to provide SI traceability. Metrology institutes are working in conjunction with the clinical community to continue to identify target analytes. Increasing collaboration between IFCC and OAWG members is a priority.

Although traceability has been a primary focus of the clinical community, many other areas are increasingly seeking out traceable primary calibrators. This is particularly true where measurements are potentially subject to review in courts of law or courts of arbitration, such as the forensic and anti-doping communities. Where testing is related to aspects such as food safety or the trade of food products, the traceability of testing results is increasingly important to reduce the risk of the emergence of regulatory and technical barriers to trade.

Assessment of capabilities for organic purity value assignment

Through collective efforts, the OAWG has greatly advanced the metrology of chemical purity analysis for organic calibrators by the sharing of best practice approaches and the resulting improvements in methodologies internationally. Activities to assess participating laboratories’ capabilities to characterize traceable organic calibrators within the OAWG have spanned nearly two decades. This program was initiated with a series of pilot (P) studies [9] which led to formal key (K) comparison exercises with the launch of the CCQM-K55 series with 17β-estradiol in 2009 [10]. Table 1 lists the relevant studies.

Table 1.

CCQM OAWG pilot (P) studies and key (K) comparisons for the determination of chemical purity of organic calibrators

Year	Comparison	Measurand	Purity, mg/g
2001	CCQM-P20.a	Tributyltin	949.2a
2002	CCQM-P20.b	o-Xylene	983.2b
2004	CCQM-P20.c.1	Atrazine	980.5a
2004	CCQM-P20.c.2	Atrazine	991.3a
2004	CCQM-P20.d	Chlorpyrifos	996.0a
2007	CCQM-P20.e.1	Theophylline	998.6b
2007	CCQM-P20.e.2	Theophylline	983.1b
2008	CCQM-P20.f	Digoxin	980.2c
2009	CCQM-K55.a	17β-Estradiol	984.3c
2010	CCQM-K55.b	Aldrin	950.8c
2012	CCQM-K55.c	L-(+)-Valine	992.0c
2013	CCQM-K104	Avermectin B1a	924.6c
2015	CCQM-K55.d	Folic acid	906.5c
2018	CCQM-K148.a	Bisphenol A	TBD

^aMedian of reported results

^bPreparative value

^cReference values assigned from the consensus data set

Over the years, these exercises have progressed from relatively simplistic determinations of the total purity to a comprehensive determination of all impurities present in an organic calibrator material at the mg/g level. Figure 1 shows that participation has steadily increased.

Fig. 1.

Participation of NMI/DIs in CCQM OAWG purity pilot (P) studies (black filled circle) and key (K) comparisons (black open circle) from 2001 to present. CCQM-K104 (red filled square) was a new area for the WG

The CCQM-K55 series of key comparisons [10, 11] covered the “purity of organics” measurement space across a 4-sector model based on molecular weight (MW, g/mol) and the log octanol–water partition coefficient (pK_OW) as a surrogate for polarity. The four sectors were defined as (MW < 300, pK_OW < −2), (300 < MW < 500, pK_OW < −2), (MW < 300, pK_OW > −2) and (300 < MW < 500, pK_OW > −2). The CCQM-K55 series was completed in 2017, and the coordinating laboratory, BIPM, created “score cards” for each participating institute that provided a graphical representation of their performance. These results have been leveraged to directly support each institute’s calibration and measurement capability (CMC) claims for pure organic calibrators as part of a demonstration of international equivalence of their SI traceability claims of their measurement standards and issued certificates through the CIPM Mutual Recognition Arrangement (CIPM MRA) [12]. This then indirectly underpins the CMC claims for the assignment of the mass fraction content of analytes present at trace levels in complex matrices.

Figure 2 displays the model that the OAWG now uses to map the organic purity space up to a MW of 1000 g/mol. This evolved model recognizes that at lower MWs the analyte polarity largely determines the analytical approach, e.g., gas chromatography (GC) for low polarity compounds versus liquid chromatography (LC) for high polarity. The extension of the MW maximum from 500 g/mol to 1000 g/mol recognizes the growing importance of more structurally complex analytes in organic analysis, such as biomarkers.

Fig. 2.

Model for OAWG purity comparisons based on different measurement approaches. Analytes in past CCQM purity studies are in bold font; chemical classes contemplated for future studies are in italics. Adapted from the OAWG strategy document [4]

The mass balance approach to organic purity

An approach using a complementary ensemble of independent measurement methods to characterize measurable impurities, historically termed “mass balance,” has been applied as a comprehensive approach for organic chemical purity analysis [13–21]. Chemical content information obtained from a wide range of analytical techniques that identify and quantify all the impurities, organic and inorganic alike, present in a material is combined and subtracted from 1000 mg/g to estimate the mass fraction content of the main component.

For organic chemical purity assignments, the confirmation of the structure of the main chemical component is the initial task. This is generally carried out using a range of modern techniques typically including mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. The careful establishment of isomeric composition and stereo-chemistry may be necessary for some applications.

The mass balance analysis of an organic calibrator nearly always involves chromatographic separation of impurities from the main component via either gas or liquid chromatography (GC or LC), depending on the physiochemical properties of the main component. Ideally, this involves application of a “universal detector,” such as flame ionization (FID), to quantify relative mass fractions of structurally related organic impurities [22]. This “organic purity” needs to be corrected for the relative mass of all chromatographically invisible impurities, including volatiles that elute with the solvent front, nonvolatiles that do not elute, water and inorganics.

In some cases, volatile solvents can be identified with GC either directly or via a headspace analysis. Increasingly, ¹H NMR is also used to identify and quantify levels of residual solvent impurity. Thermal gravimetric analysis (TGA) has been employed as a routine method to measure all volatile impurities including water, although reliance solely on TGA has been problematic when solvents—especially water—are entrapped within the solid crystalline form. To address this, specific water analysis via Karl Fischer titration has become more routinely employed.

When applying any measurement technique, it is important to understand the limitations of that technique. This is particularly true for chromatographic techniques selected for their applicability to the main component, which may not translate to applicability for all impurities of “related” structure. For example, higher MW impurities may not be as amenable to GC as the main component. The detector of choice may also be limited. Photodiode array detection, the most commonly applied detection method for LC amenable analytes, will only detect compounds with a UV chromophore. As an example, a significant discrepancy on the order of 10 mg/g for the assessment of the mass fraction of aldrin in the CCQM-K55.b comparison was observed between most mass balance results and those obtained using quantitative NMR (qNMR) methods [11, 23]. Subsequent analysis conducted by the National Metrology Institute of Japan (NMIJ) using size exclusion chromatography identified a high MW impurity that accounted for this difference. Such methods were not employed by NMI/DIs routinely for purity analysis in 2010 and are still not regularly employed in our laboratories today. However, the results from this comparison reinforced two findings: (1) the mass balance method can underestimate purity if all impurities are not adequately accounted for, and (2) methods such as qNMR, if conducted expertly, provide a direct measure of the main component and perform a valuable role as a check and independent confirmation of results obtained by the classical mass balance approach.

The lessons learned from these comparisons have led to better assessment of the full range of possible impurities using a wide range of techniques (Table 2). Most institutes try to obtain supporting evidence for all their detected impurity quantifications. For inorganic residues, for example, many institutes utilize inductively coupled plasma (ICP) approaches in addition to TGA. ¹H NMR is now routinely used to measure the mass fractions of minor impurities such as solvents, as well as directly assign the mass fraction of the main component via utilization of a traceable internal standard. Table 2 lists the commonly used analytical techniques.

Table 2.

Typical techniques for the determination of chemical purity of organic calibrators

	Measurand	Liquid chromatography (LC)	Gas chromatography (GC)	Headspace GC	Thermogravimetric analysis (TGA)	Ion chromatography	Inductively coupled plasma-mass spectrometry and/or optical emission spectrometry (ICP- MS/OES)	X-ray fluorescence (XRF)	Size exclusion chromatography (SEC)	Titrationa	Coulometry	Differential scanning calorimetry (DSC)	Nuclear magnetic resonance (NMR)b
Indirect	Structurally related organics	✓	✓										✓
	Solvents		✓	✓	✓								✓
	Water				✓					✓
	Inorganics, metallic						✓	✓
	Inorganics, nonmetallic					✓	✓	✓
	Non-volatiles, polymeric residues								✓
Direct	Primary component (direct)									✓	✓	✓
	Primary component (ratio)												✓

^aIncludes classical and Karl Fischer titration

^bIncludes quantitative NMR with external and internal standard

The determination of related structure impurities has been an area of focus for the OAWG as obtaining SI traceable estimates for the individual components of this class of impurities is challenging. One approach is for each structurally related impurity identified within a material to be quantified via chromatographic techniques using a calibrator for the impurity. This is not possible in all cases, and thus, the estimate of each impurity needs to be determined by converting the chromatographic peak area to a relative mass fraction for the impurity. In GC-FID analysis, the main consideration will be the percentage of carbon in the impurity compared to the main component and the respective functionality. For LC-UV, the respective chromophores and molecular weights need to be taken into account. This requires two steps to be achieved effectively: (1) the measured peak area needs to be converted via the relative response factor of the impurity versus the main component to give a relative mole fraction, and then (2) the mole fraction needs to be converted to a relative mass fraction using the impurity’s MW. In the numerous cases where the λ_max of the impurity and/or its chemical structure is considerably different from that of the main component, if the relative response factor is not assessed appropriately then the level of the individual impurity can be considerably over or underestimated. Assessing the best estimate of the MW, for example, via mass spectrometric assessment, is also required for conversion from chromatographic peak areas to relative mass fractions. Without careful assessment of these factors, the traceability of impurity values for individual structurally related impurities is not achieved. This can then lead to lack of traceability in the mass fraction assessment of the main component [14].

A complete and comprehensive review of the general indirect and direct approaches to chemical purity assignment for organic calibrators will be provided in the IUPAC Organic Purity Technical Report “Methods for the SI Value Assignment of the Purity of Organic Compounds (IUPAC Project 2013–025)” currently in review [24]. This report is being prepared by experts within the chemical metrology community, including many OAWG members, and captures best practice approaches at this time.

Direct purity assessment techniques

Traditional primary direct methods [1, 2, 25], such as coulometric titration and differential scanning calorimetry (DSC), have been employed to achieve direct measurement of mole fraction content of organic materials. These methods are appropriate only for very pure materials. Acidimetric titration is limited to analytes with an exchangeable proton. DSC was the most commonly employed direct purity technique when the OAWG commenced purity comparisons, but it was considered to have been applied inappropriately on occasion and has fallen out of favor. Currently, these techniques are used in a limited number of institutes who have maintained specialized capabilities.

Quantitative nuclear magnetic resonance (qNMR) spectroscopy is a routine primary ratio method that is increasingly used for the direct determination of the main component in organic purity analysis [26, 27]. Initial work on its application as a primary method began many years ago, and the early results reported by the few pioneer institutes were unacceptably variable [28–30]. In the recent CCQM-K55.d folic acid comparison, 12 of the 18 participants either reported values that involved qNMR or else separately reported a mass fraction value exclusively via qNMR [31]. Although folic acid has significant solubility challenges and is labile once in solution under certain conditions, making it a particularly challenging analyte for qNMR, the dispersion of qNMR results was only slightly wider than for the mass balance results. In general, continual increase in the use of qNMR and demonstration of its proficiency has been seen throughout the decade of CCQM-K55 series activities.

The OAWG has also invested significant effort in a series of recent pilot studies (the CCQM-P150 series) to examine in detail potential biases associated with the implementation of qNMR as a primary method. Fifteen metrology institutes participated in the most recent CCQM-P150.b study. What has been revealed is that the qNMR method is not impervious to bias; however, the best practice application of qNMR continues to develop within the OAWG membership. As with any primary method, metrological rigor is required and reports by the coordinating laboratory (NMIJ) on the results of the CCQM-P150 pilot studies outline critical aspects related to sample preparation, instrument setup and data analysis [32]. The topic of measurement uncertainty was carefully assessed in the recent CCQM-P150.b study, and it revealed inconsistencies in approach and distinct cases of underestimation. Measurement uncertainty evaluation is a critical factor that remains a priority area for the OAWG.

Although further work is needed to harmonize best practice uncertainty estimation in conjunction with the application of qNMR, it has proven to be an invaluable tool in organic purity assessment and is now commonly employed as a stand-alone technique [33, 34]. When combined with mass balance approaches, it constitutes a powerful and reliable method for purity assignment.

The combination of purity assessment results

In general, OAWG members have significantly evolved how they handle assessment of chemical purity and the associated measurement uncertainty, often through combining information from both the mass balance and qNMR techniques [35–38]. Many members now routinely apply both mass balance and qNMR to allow more effective assessment of bias in the purity assignment [39], or “dark uncertainties” [40], that are discernible from differences between methods. Both mass balance and qNMR approaches can have biases that are difficult to infer independently without considerable effort. With the mass balance approach, important questions to consider include “have all impurities present in the material been detected?” and “are any observed impurity signals actually artifacts inherent in the measurement method?”. For qNMR, any interference on the analyte or internal standard signals will bias the result; even for moderately complex compounds, this can become an issue. A wide range of other factors related to sample preparation and data acquisition need to be effectively controlled to produce traceable purity estimates. Conducting both mass balance and qNMR methods in parallel is now widely utilized to provide independent purity estimates that can be combined for a comprehensive assessment of chemical purity. This should always be carried out in combination with the critical technical evaluation of the results; however, the use of multiple techniques, while not failsafe, is an excellent approach for the assessment of bias.

The National Institute of Standards and Technology (NIST) has led development of Bayesian models for estimating chemical purity with realistic uncertainty estimates for qNMR methods [41], and for combinatorial approaches that leverage both mass balance and qNMR methods [42, 43]. Figure 3 illustrates examples of the latter approach for a range of scenarios from when both mass balance and qNMR methods agree (top panel) to when an irreconcilable difference exists between the methods, but the qNMR results are trusted over the mass balance results (bottom panel; scenario b). A demonstration of the latter was recently reported [43] for the NIST determination of 3-epi-25(OH)D₃ consensus purity values in which the mass balance approach did not fully account for all sources of impurities and was thus deemed biased high relative to the qNMR results. Though there is greater confidence in the qNMR method result for this example, there was not unequivocal technical evidence that the mass balance method is the source of apparent bias. The approach presented in Fig. 3 estimates the likelihood of all possible “true” values through utilization of all available data and analyst expert insight. Such an approach is easily managed with Bayesian estimation models.

Fig. 3.

Examples of the use of probability distribution functions (PDFs) to determine consensus purity values over three cases where there is good agreement between qNMR (blue solid line) and mass balance results (red solid line) (top panel), a more common scenario of partial overlap between results (middle panel) and poor agreement between results (bottom panel). The PDFs marked as “a” (black dotted line) simply utilize the mean values from each approach, whereas the PDFs marked “b” (green dotted line) use a qNMR-favored distribution for the Bayesian assessment of purity

An important feature of this approach is the realistic assessment of measurement uncertainty associated with the individual results of the specific techniques. This is a critical first step before data from multiple methods should be combined.

Challenges

As the application of metrology for organic chemical measurements has evolved, so has the need to extend the capability of pure calibrator certification into measurement sectors that tend to merge with the bioanalytical sciences. The OAWG’s recent experience with CCQM-K104, a key comparison specifically coordinated to examine the assessment of purity for larger natural products, revealed the increased challenges associated with the presence of isomers and homologues that are common with chemical products isolated from natural sources. The analyte chosen in this case was avermectin B1a with a MW of 873 g/mol. Avermectins are a class of naturally occurring biological pesticides that are generated from microbiological fermentation processes. The biosynthesis of compounds of increasing size and complexity typically is associated with the production of impurities, many of which may be isomers or homologues of the main component and are difficult to remove during purification [38]. The ability to chromatographically resolve each impurity and effectively quantify them presents a significant challenge. Furthermore, for the direct analysis of such compounds via qNMR it becomes increasingly difficult to take into account contributions from similar analogs that may not always be fully resolved from the quantification peak for the main compound. Several institutes are addressing these challenges by investigating more sophisticated approaches to qNMR such as combining chromatography with NMR and utilizing 2D NMR techniques adapted for quantification. The OAWG is committed to the demonstration of capabilities for larger molecule purity assessment and to the consideration of advancing approaches to deal with our emergent measurement challenges [44, 45].

Recognition of capabilities

The primary organic calibrator services provided by metrology institutes continue to expand. The fundamental capability of assigning SI traceable purity values to organic calibrators is a basic expectation for all participants in the OAWG. As a result, 18 institutes formally demonstrated these capabilities in the most recent comparison CCQM K55.d folic acid [31]. Over 450 approved CMCs in the CIPM Mutual Recognition Arrangement [12] database for pure organics are now effectively supported through participation in the CCQM-K55 comparison series. Furthermore, the number of pure organic material CMCs has risen steadily. Most of these CMCs currently recognize capabilities for institutes to produce specific CRMs for individual chemicals. The evidence from the many years of purity key comparisons coordinated by the OAWG has meant institutes are now able to claim broader capabilities. With the introduction of such broader scope CMCs that cover much larger numbers of compounds within a single entry, the total CMC numbers may reduce; however, the overall scope of services covered will substantially increase. Broader scope CMCs are expected to be presented in a range of ways, such as coverage for an entire class of chemicals (e.g., for all organochlorine pesticides). Alternatively, they may be more generic and aligned to the scopes of key comparisons. For OAWG purity comparisons, an example broad scope CMC could cover “polar organic compounds in the molecular mass range 100–500 g/mol.”

This represents a significant shift in the presentation of CMCs, and it has taken many years of comparisons of pure organics and the commitment of various institutes to reach the point where there is now confidence within the OAWG to promote and accept broad CMCs. It demonstrates that the OAWG now understands the issues associated with the key techniques that are employed and has evidence from a decade of CCQM-K55 comparisons to show how institutes have benchmarked their abilities to deal with these issues and learn from each comparison. To maintain support for broad claim CMCs, the OAWG will require an ongoing program of key comparisons examining the value assignment of pure organic calibrators. The OAWG is committed to this as a priority area and to the continued assessment and development of best practice techniques for organic chemical purity assessments.

Conclusions

The OAWG activities over the last two decades have led to significant improvements in approaches by metrology institutes in the certification of organic calibrators. The application of a combination of direct and indirect approaches provides a best practice methodology that most effectively assesses potential biases. The organic purity assessment capabilities maintained by national metrology institutes ensure that CRMs comprising the pinnacle of SI traceability chains are internationally recognized and are internationally comparable. Many of the chemical CMCs in the KCDB are related to pure organic calibrators, and there are similarly many entries in the JCTLM database, highlighting the importance of these primary calibrators to the laboratory medicine community. These capabilities also underpin metrology institutes’ programs producing calibration solutions and matrix-based CRMs, as these also rely on the availability of traceable pure organic calibrators. Following the release of documents such as ILAC P10, the broader chemical community has become more aware of the need to ensure the metrological traceability of testing results and the importance of higher-order pure organic calibrators continues to grow.