Mass Balance Method for the SI Value Assignment of the Purity of

Article pubs.acs.org/ac

Mass Balance Method for the SI Value Assignment of the Purity of Organic Compounds Steven Westwood,* Tiphaine Choteau, Adeline Daireaux, Ralf Dieter Josephs, and Robert Ian Wielgosz Bureau International des Poids et Mesures (BIPM), Pavillon de Breteuil, F-92312 Sèvres Cedex, (33) 1 45 07 70 57, France S Supporting Information *

ABSTRACT: A mass balance method is described for determining the mass fraction of the main component of a high purity organic material. The resulting assigned value is established to be traceable to the SI and can be determined with a small associated measurement uncertainty. Pure organic materials with values and uncertainties determined in this way are necessary as primary calibrators of reference measurement systems in order to underpin the metrological traceability of routine measurement results. The method has been applied to materials in which the main components were respectively theophylline, digoxin, 17βestradiol, and aldrin. Its performance has been validated in international comparisons coordinated by the BIPM and is in principle applicable to a wide structural range of stable, nonvolatile organic compounds. It has been successfully applied to mass fraction assignments when the main component is present in the range of (950−1000) mg/g and can achieve associated standard uncertainties ranging from 0.5 mg/g (for high purity materials or those containing well-characterized, stable minor components) to 2 mg/g (materials with a significant number or variety of impurities). It is in principle equally applicable to materials with a smaller mass fraction content of the main component.

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ncreasingly, compliance with international regulatory1−3 and accreditation 4 requirements places emphasis on the reliability and quality of routine chemical measurements in organic analysis. This has in turn led to the need to clearly identify how the metrological traceability and measurement uncertainty of these results are established. For the measurement of all well-defined organic species in a matrix to be metrologically traceable to the International System of Units (SI) system requires the measurement method to be validated to furnish valid results within a stated uncertainty. In the majority of cases this traceability to the SI is ultimately achieved through linkage to a calibrator material of the defined species, the purity of which has been established in a manner that is also traceable within a stated uncertainty to the SI.5 It should be emphasized that linkage to a suitable primary calibrator is a necessary but not sufficient condition to establish the traceability of the result of a measurement procedure, and it is ultimately the responsibility of the measurement service provider to validate the traceability of the results obtained using their procedure. Agreement on what constitutes a valid method to measure the purity of a nominally pure material within a stated © 2013 American Chemical Society

measurement uncertainty is needed. The purpose of this paper is to describe the approach, performance, and applicability of the mass balance method implemented for this purpose at the BIPM and validated in international comparisons to assign organic purity. Although the performance of the approach is only described for a limited number of compounds, with appropriate validation it is applicable in principle to a wide range of stable, nonvolatile organic compounds with typical associated standard uncertainties in the resulting assigned values from 0.5 mg/g (for high purity materials or those containing well-characterized, stable minor components) to 2 mg/g (for materials with a significant number and/or variety of impurities). Simplification of the method is also possible provided the resulting increase in measurement uncertainty is properly accounted for. The materials whose mass fraction assignments are described in this paper were characterized in the course of international comparisons of organic purity assignment capabilities underReceived: October 31, 2012 Accepted: February 13, 2013 Published: February 13, 2013 3118

dx.doi.org/10.1021/ac303329k | Anal. Chem. 2013, 85, 3118−3126

Analytical Chemistry

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approach encompasses techniques providing a direct measure of the main component (gravimetry, titrimetry) or to establish it by comparison to a standard of the same quantity (quantitative NMR, stable isotope ratio mass spectrometry, relative chromatographic response). Each approach relies on the validity of fundamental assumptions applicable to the method and are open to significant bias in the case that these base assumptions do not hold. The approaches taken to address these potential biases for the mass balance approach is one of the subjects of this paper. Additionally, the standard uncertainties associated with the results of assay techniques that require external calibration to quantify the main component are relatively large (typically on the order of (1−2)% relative to the mass fraction of the main component). These are significantly larger in absolute terms than those routinely achievable using the mass balance method described herein.

taken to enable National Measurement Institutes (NMIs) to demonstrate their measurement capabilities in the area of organic analysis, and their basis for metrological traceability. The comparisons involved the purity assignment of samples of the following compounds: • Theophylline (1) (high purity and spiked with caffeine and theobromine)6 • Digoxin (2)7 • Estradiol (3)8 • Aldrin (4)9 In the discussions below, the reference values used to validate the performance of the BIPM approach for mass fraction assignment refer to the best estimates for the content of the main component and its associated impurity profile in each comparison material. These reference values were derived from and agreed to by peer review of the combined participant data.

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CONCEPT OF PURITY AS APPLIED TO ORGANIC CALIBRATORS In the context of organic analysis, the ambiguous concept of “purity” is generally realized and reported in terms of the mass fraction of the main component present in a nominally pure material. The simple mathematical description of the mass fraction of the main component of a pure material is shown eq 1. mA wA = mA + ∑ mx (1)

BIPM MASS BALANCE PURITY ASSIGNMENT METHOD For a given organic compound, the individual classes of impurity that generally require assessment and quantification are the following: (1) related structure impurities (wRS) (2) water (wW) (3) residual organic solvent (total volatile organic compounds) (wOS) (4) nonvolatiles (wNV), with potential contributions from either or both: (a) inorganic impurities (b) nonvolatile organics When the mass fraction assignments of impurities are orthogonal and cover the total impurities present in the material, eq 2 assigns wA where all components are in units of milligrams per gram:

Where, wA = mass fraction of the main component A in the material, mA = mass of A in a gravimetrically defined aliquot of the material, ∑mx = combined mass of 1, 2, 3, ..., x minor components (impurities) present in the aliquot. It can be expressed in the SI units of kilogram per kilogram but for numerical convenience and for the purposes of this paper, wA is reported in units of milligram per gram (mg/g). The upper limit for the mass fraction of the main component, equivalent to “100 % purity” in the common but ambiguous usage often found in the general literature, is thus 1000 mg/g.

wA = 1000 − (wRS + wW + wOS + wNV )

(2)

Equation 3 describes how the combined standard uncertainty associated with wA is obtained by quadratic combination of the uncertainties associated with each contributing impurity assignment:

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APPROACHES TO THE ASSIGNMENT OF MASS FRACTION PURITY Estimates of the mass fraction content of the main component A in an organic material which contains x independent minor components (impurities) are obtained in general using one or more of three general approaches:10 (i) determination of the mass fraction (w1, w2, ..., wx) for each impurity in the material followed by subtraction of the summation of impurities from the limit value; (ii) determination in one measurement of the combined mass fraction (∑wx) content of the impurities in the material followed by subtraction from the upper limit value; (iii) direct determination of the mass fraction of the main component. The first approach is variously referred to as the “mass balance” or “summation of impurities” approach, and our implementation of this approach is the subject of this paper. The second approach utilizes thermal methods (such as differential scanning calorimetry and adiabatic calorimetry) to measure phase-change phenomena of a material, the results of which are related to the total impurity content. The third

u(wA ) =

u(wRS)2 + u(wW )2 + u(wOS)2 + w(wNV )2 (3)

In the following sections the approaches used at the BIPM to quantify each class of impurity are summarized, with a description of key challenges that have arisen and been addressed in the course of developing and implementing the method to characterize specific compounds. Measurement of Mass Fraction of Related Structure Impurities (wRS). Experimental Method and Description. High resolution gas chromatography (GC) and liquid chromatography (LC) techniques coupled to detectors with broad selectivity and high sensitivity were used to detect and quantify this class of impurity. The detection systems used at the BIPM to deliver the mass balance purity assessments described in this paper were diode-array UV detector (UV) and MS/MS detection using electrospray (ESI), atmospheric pressure chemical ionization (APCI), or atmospheric pressure photoionisation (APPI). More recently, use of LC coupled to a charged aerosol detector (CAD)11 has also been incorporated into our procedures. For GC methods either a flame ionization 3119



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GC-MS using electron impact ionization and confirmatory quantification by GC-FID, to perform the related structure impurity quantifications for this material. It is necessary to control for the potential formation of artifacts in the course of sample preparation and chromatographic analysis. These could otherwise be mistaken for genuine impurities. This problem arose specifically in our analysis of estradiol and its resolution is discussed in detail below. By developing independent chromatographic conditions capable of resolving the main component from longer and shorter retention time potential impurities, greater confidence was obtained that, when applied to the material to be characterized, coelution of related impurities with the main component was unlikely to be a significant issue. The BIPM approach also assumes that if examination of the material by high resolution chromatography on independent columns using different detectors failed to identify any significant additional organic components, and this is confirmed by NMR and is consistent with the results of elemental analysis (noting the limitations of these two latter techniques: 1H NMR relies on sufficient resolution of each component, and elemental microanalysis can only identify bias if the undetected impurities are of significantly different empirical formulae (C, H, N %) to the main component), then it was assumed that no further “nondetectable” related structure impurities were present in the material and that no further uncertainty component was required to cover this assumption. Metrological Uncertainty and SI Traceability of Mass Fraction of Related Structure Impurities. Use of the external calibration approach to quantify minor impurities using LCMS/MS quantification allows for sensitive and selective quantification of minor impurities in the presence of a structurally related major component. We have found that the relative standard uncertainty achievable in the mass fraction assignment of minor components are comparable to those that can be obtained for the main component under the same circumstances. They are typically in the range (1−2)% for the quantification of minor components present above the 10 mg/g level and (2−5)% for compounds present in the range (1−10) mg/g. Larger relative uncertainties are associated with the quantification of impurities present at levels below 1 mg/g, but these do not make a significant contribution to the overall absolute standard uncertainty for the derived value for the main component. These levels of measurement uncertainty in the individual impurity quantifications of the minor components generally result in a significantly smaller absolute uncertainty in the mass fraction of the main component assigned through the mass balance approach than those associated with direct assignment of the main component by external calibration against a reference standard. To establish SI traceability of the results for the total related structure impurities, it is necessary to demonstrate that the result can be related, within associated uncertainty, to stated references through an unbroken chain of calibrations.14 In this context the stated references are the individual reference materials used to calibrate each external measurement. In the majority of cases, the structure of the impurities was established and a reference material was available for each impurity component. In these cases, the traceability of the mass fraction for each impurity was to the corresponding reference material used for the external calibration. In the smaller number

detector (FID) or mass spectrometry (MS) with electron impact (EI) ionization were used. The purity assignment for each compound commenced with the gravimetric preparation of a synthetic test solution consisting of a mixture of the main component and a range of potential related structure impurities. Two separate chromatographic systems capable of resolving the components of this test mixture were then developed. The aim was to achieve baseline separation of each of the components under the conditions used, though if this was not feasible, the minimum requirement was baseline separation of the main component from all other potential impurities. The performance characteristics for each method for each component (LOD, LOQ, sensitivity) were determined over a mass fraction range representative of the levels the impurity could potentially occur in the material under assessment. Optimising chromatographic separation and determining performance characteristics on a test solution rather than commencing with the material itself introduced a larger initial workload, but there were significant advantages in this approach. It enhanced confidence that all impurities associated with the main component will elute within an appropriate retention time window and will be resolved from the main component of the characterized material. In addition determining the quantification parameters and response factors, relative to the main component, of a range of impurities underpins fit-for-purpose identifications and quantifications in cases where unidentified impurities were present in the target material. One chromatographic method utilized a “universal detector” that was intended to respond to every organic component in the column effluent12 with a readily modeled response factor. This method was used to separate, detect, and, where possible, identify each related structure impurity present in the material under examination. Methods using GC-FID and more recently LC-CAD are generally regarded as meeting the requirements for universal detection of organic analytes. LC-UV methods cannot serve this role, as any impurities that lack a UV-active chromophore will by definition not be detectable. The second chromatographic method utilized a mass spectrometric (MS) detector to confirm the identity and quantify directly by external calibration every related structure impurity present in the material for which as a minimum an independent sample of confirmed identity and assessed purity was available. LC-MS/MS approaches were used for the purity assignments of theophylline, digoxin, and estradiol, and GC-MS was used for aldrin. Quantification by external calibration by LC-MS/MS was used to assign the mass fraction of each individual related structure impurity present in theophylline, digoxin, and estradiol for which a comparison material was available, where “comparison material” includes not only materials supplied in compliance with the full ISO requirements for designation as “reference materials” but also compounds of stated structure obtained from commercial suppliers, in the cases where the latter was the only available choice. The overall process has been described in detail for digoxin.13 Where a comparison material was not available for a specific impurity and thus quantification by direct external calibration was not possible, an estimate was made assuming a calibration response consistent with that of the closest related structure impurity for which comparison material was available. For aldrin we were not able to achieve LC ionization conditions using ESI or APCI techniques and relied instead on 3120



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Figure 1. Degree of equivalence of BIPM mass balance estimates with reference values for each impurity subclass for each assigned material where a1 = high purity theophylline;6 a2 = spiked theophylline;6 b = digoxin;7 c = estradiol;8 d = aldrin.9 The error bars show the expanded uncertainty (Ui) associated with each Di.

used was to assign the mass fraction of the material (x) using eq 4 with values expressed in units of milligrams per gram:

of instances where an impurity could not be identified or it was identified but no reference material was available, traceability was to the selected reference material(s) of related structure impurity(ies) whose calibration response was the basis for the value assigned to the unknown impurity. In these latter cases the uncertainty in the assigned value was expanded appropriately and conservatively to reflect the lack of a direct external calibrant. Thus traceability requires a fit-for-purpose, SI-traceable mass fraction assignment for each pure substance reference material used as the basis for each external calibration. There are three potential cases applicable to the source of the materials used for external calibration. They can be obtained as follows:

x = xmin +

1000 − xmin 2

(4)

In such a case, and in line with literature recommendations,15 the standard uncertainty in the assigned value (ux) assumes a rectangular distribution of possible values between the lower and upper limit values and is given by the numerical eq 5 expressed in milligrams per gram: ux =

1000 − xmin 2 3

(5)

The extent of the additional characterization studies on impurity reference materials undertaken in-house were a function of the absolute amount of the impurity in the pure material being characterized. When the impurity is present at levels below 1 mg/g, even a large bias in its value as assigned using eqs 4 and 5 will not make a significant contribution, in the context of the mass fraction of the main component, to the absolute value of the combined related structure impurity content or its standard uncertainty. Traceable results are assumed to be obtained from external calibrations based on the provider’s reported purity for these materials provided chromatographic analysis of the material is consistent with the assigned value. When the impurity is present at higher levels further in-house checks, which include some or all of thermogravimetric analysis (TGA),1H NMR, or elemental analysis in addition to the standard requirement for a confirmatory chromatographic analysis are undertaken to the extent required to provide sufficient confidence in the validity of the value assigned using eqs 4 and 5. It is quite possible that the additional data obtained by these additional characterization studies could be used as the basis for a more accurate value assignment than those given by application of eqs 4 and 5, but in practice this would have no significant impact on the final mass balance value for the main material. Representative Examples of Determination of Total Related Structure Impurities. Total Related Structure Impurities (wRS) in “Spiked” Theophylline. A purity assignment was carried out on a spiked theophylline (1, Figure 1) containing gravimetrically assigned amounts of caffeine and

(1) Certified reference materials prepared in compliance with ISO Guide 34 requirements and with assigned mass fraction values traceable to the SI; (2) Commercially available materials supplied with a certificate of analysis providing varying degrees of information on the characterization and purity of the substance, often providing a limit value for the purity of the material; (3) Uncharacterized materials with no assigned purity. Case 1 is clearly the simplest and would require no additional supporting investigation. Unfortunately this is always going to be a rare situation and was not encountered in the investigations described in this paper. In practice, all the studies reported in this paper involved reference materials corresponding to case 2, and this is likely to be the case in general for purity assignments. Additional characterization was carried out at the BIPM on the impurity reference materials to determine an appropriate value for the mass fraction of the main component in this material and its associated uncertainty that would encompass its true value. Case 3 would require an in-house investigation to a degree sufficient to assign the purity in a fit for purpose manner and again would be dependent on the amount of impurity present in the material. In practice we have not encountered this situation to date. Where a material from a commercial supplier was reported to have a minimum purity value of xmin mg/g and this value was consistent with additional BIPM measurements, the approach 3121



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theobromine.6 Two independent LC methods, using different columns, solvent systems, and detectors (respectively UV absorption at 273 nm and MS/MS with positive ESI) were validated using the common xanthines theophylline, theobromine, caffeine, 3-methylxanthine, and 7-methylxanthine as model compounds. Application of both methods to the sample, with quantification by external calibration for each identified minor component, and quantification from the relative area response for one unidentified impurity, produced after combination of the data a related structure impurity profile for the material as provided in Table 1 of the Supporting Information. The wRS assigned by the BIPM was 13.9 mg/g with a combined standard uncertainty of 0.55 mg/g. This agreed within its associated uncertainty with the reference value for wRS of 13.4 mg/g with standard uncertainty of 0.6 mg/g assigned from the combined participant data.6 The identity and the relative amounts of the individual impurities also agreed with the data reported by other participants. Suitable traceability to the SI for this value was established by confirming that the values on the Certificates of Analysis provided for the reference materials used for the individual quantification of the impurities caffeine, theobromine, and methylxanthine met the acceptance criteria described above in Metrological Uncertainty and SI Traceability of Mass Fraction of Related Structure Impurities. The mass fraction of an unidentified, UV-active impurity also present in the material was assigned based on the sensitivity factor and UV-response factors of the reference materials for the other impurities present. A large type B uncertainty was given to the assigned value to ensure that the overall claim for traceability to the SI of the combined related structure impurity estimate for theophylline was justified. Total Related Structure Impurities (wRS) in Estradiol. The assignment of wRS in estradiol was initially undertaken using separate LC-UV and GC-MS methods. The LC-UV method used detection and quantification of absorbance at 225 nm while the GC-MS quantification used total ion monitoring of a bis-silylated derivative and electron impact (EI) ionization. Both methods identified and gave consistent values for the main impurities (4-methylestradiol, estrone, and estratriol). Some additional impurities seen by LC-UV were not confirmed by GC-MS but it was assumed these were polar, polyoxygenated steroids and were unstable under the derivatization conditions used for GC. This assumption proved to be incorrect (vide infra), and as a result, the first implementation of the LC-UV method resulted in an overestimate7 for the related structure impurity content as the additional peaks in the LC-UV analysis resulted from an undetected artifact formation by in situ oxidative coupling of estradiol in the neutral LC sample solutions prior to injection.16 In follow-up studies, a normal phase LC-MS/MS method using APPI and isopropanol/hexane mixtures as solvent was validated and shown to avoid artifact formation. It was also demonstrated that artifact formation in the LC-UV method was suppressed when the aqueous sample solutions were adjusted to low pH.17 The full related structure impurity profile assigned to the estradiol material using these revised methods is set out in Table 2 of the Supporting Information. The value for the mass fraction of related structure impurities obtained by the revised method was 8.96 mg/g with a standard uncertainty of 0.43 mg/g, in agreement with the reference value

assigned to the material8 of 8.65 mg/g with a standard uncertainty of 0.16 mg/g. Measurement of Mass Fraction of Water (wW). Experimental and Method Description. The Karl Fischer (KF) titration method is the principle source of information on the water content of all organic compounds characterized by the BIPM.18 Direct addition to the titration cell or transfer of the water content from a solid sample via a heated sample oven were used as appropriate. When the heated sample oven transfer was used the influence of oven temperature was investigated to establish the minimum temperature for release of water from the sample. The water content estimate obtained by KF titration must also be consistent with results obtained by TGA and elemental analysis. In a situation where heated sample oven KF titration complemented by TGA is used, the potential for both results to be biased due to the elimination of water from the material should be assessed. In this case it would be particularly important for the overall mass balance value for main component purity to be cross-checked by an independent method such as qNMR. Equation 6 is used in our calculations of water content by Karl Fischer titration: ⎛ R KF − R blank KFblank ⎞ wW = ⎜ x x ⎟ massx ⎝ ⎠

(6)

Where, Rx = recovery factor for water content transfer from sample x, KFx = KF titration water content (in milligrams) for sample x, Rblank = recovery factor of water content transfer from blank, KFblank = KF titration water content (in milligrams) for blank, massx = net mass (in grams) of sample x Traceability and Uncertainty (u(wW)) of the Mass Fraction of Water. The Karl Fischer method was operated as a potentially primary method, where the coulometric value measured by the titrator was converted into an equivalent amount of water, and the assumptions behind this and the traceability to the SI of the water content value measured by the titrator was validated by demonstrating its unbiased operation when a water saturated octanol certified reference material was used as a check sample. When it was not possible to distinguish a statistical difference between the results for the sample and blank vials, the material was assigned a water content of zero with an associated positive uncertainty equivalent to the limit of detection. For the KF titration apparatus using oven transfer used to undertake these measurements, a minimum absolute amount of 100 μg of water was needed in the sample to be able to distinguish between the variation of blank measurements and the presence of water in the material. This corresponds to a level of water content of 0.1 mg/g when a 100 mg sample was used for individual analysis. The positive standard uncertainty associated with a limit value assignment of 0 mg/g water content was 0.14 mg/g, corresponding to three times the standard deviation of blank sample repeat measurements. This uncertainty associated with water content determination is one of the limiting factors in the measurement uncertainty achievable for main component assignment by our mass balance method. When direct addition is used, the background level is lower and smaller sample sizes can be used. The absolute limit of detection for water content was 10 μg (corresponding to a minimum sample size of 10 mg if water content is 0.1 mg/g) for individual analysis. Representative Examples of Determination of Water Mass Fraction. Mass Fraction of Water (wW) in Estradiol. For the 3122



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estradiol material,8 the titration was performed by direct addition of aliquots of the sample into the cell and titration of the water released on dissolution of the solid into the anhydrous reagent. The blank value was established by determination of the adventitious addition of water into the cell during dummy additions. The water content assigned by KF titration in this manner was consistent with TGA data and the elemental analysis results for the material. The mass fraction of water in estradiol reported by the BIPM was 7.5 mg/g with a standard uncertainty of 0.44 mg/g, which agreed within their respective uncertainties with the reference value of 6.75 mg/g with a standard uncertainty of 0.21 mg/g established for the material. Mass Fraction of Water (wW) in Pure Theophylline. For the water content determination of the high purity sample theophylline,6 direct addition was not possible due to the lack of solubility of the compound in the KF titration reagent. The assay was performed instead using the transfer oven method. The result was consistent with results from TGA and elemental analysis. The mass fraction of water assigned to the material was 0.2 mg/g with a standard uncertainty of 0.1 mg/g, which agreed with the reference value assigned for the material of 0.2 mg/g with a standard uncertainty of 0.05 mg/g. Measurement of the Mass Fraction of Residual Organic Solvent (wOS). Experimental and Method Description. Residual solvent content (wOS) was determined by direct injection of a solution of the material onto a capillary GC column suitable for analysis of volatile organic solvents, with the column outlet connected to a mass selective detector. Two independent solutions were analyzed, one in a volatile solvent such as methanol and the other in a relatively nonvolatile solvent such as DMSO or DMF. The difference in retention time for the solvent peaks between the two solutions controls for the possibility of residual solvent obscured by one or other of the sample solvent peaks. When residual solvent was detected, it was quantified by a GC-MS method using external calibration against a reference material for each residual solvent and with an internal standard to control for analyte volatility. 1 H NMR was used to verify the identification and the quantification of any significant solvent residue in the material. Traceability and Uncertainty of the Mass Fraction of Residual Solvent. The measurement uncertainty and traceability to the SI of the value for wOS involves essentially the same issues as those for related structure impurity content as discussed previously. In practice, the use of HPLC grade pure solvent as the external calibration standard has been sufficient to meet these requirements in the determinations undertaken to date. Representative Example of Determination of Residual Solvent Mass Fraction. Mass Fraction of Residual Solvent (wOS) in Digoxin. The quantification of residual solvent in digoxin7 was undertaken using GC-MS and confirmed by 1H NMR. The digoxin material was estimated to contain ethanol (2.5 mg/g), dichloromethane (1.0 mg/g), and toluene (0.1 mg/g) giving a combined residual solvent content of 3.6 mg/g with an associated uncertainty of 0.2 mg/g. This agreed with the reference value for the material of 3.9 mg/g with an uncertainty of 0.36 mg/g and with the individual identified components. Measurements of the Mass Fraction of Nonvolatile Materials (wNV). Experimental and Method Description. The amount of nonvolatile impurities were assessed by combination

of data from several complementary methods. The inorganic content of the compound was determined by ICP-MS, allowing for individual determination of levels of the common metallic elements (Na, Mg, Al, Si, Fe, K, Ca). In no material examined to date have these been present at levels above their individual limits of detection which are in the range (20−50) μg/g. In each case the microanalysis was also consistent with the formula of the main component, and there was no significant mass residue remaining after oxidative combustion of the material at high temperature in the TGA, the results for nonvolatile residue determinations for the material were indistinguishable from blank analyses and the wNV was assigned a value of zero with an associated positive uncertainty of 0.28 mg/g, equivalent to a conservative estimate of the combined limits of detection for the ICP-MS and TGA analysis. To control for oligomeric organic impurities, it had been initially assumed that if present in significant amounts they would deviate the C, H, N microanalysis and should also be visible in the 1H NMR spectrum. While this assumption held for theophylline, digoxin, and estradiol, it proved not to be the case for aldrin8 (see Mass Fraction of Nonvolatile Residue in Aldrin below). We have incorporated gel permeation chromatography into our ensemble of characterization methods for ongoing purity assignments, specifically to control for oligomeric organic impurities that are not detected by other means. Representative Examples of Nonvolatile Residue Mass Fraction. Mass Fraction of Nonvolatile Residue in Digoxin. Application of the suite of methods described above (TGA, elemental analysis, ICP-MS) to the digoxin material7 indicated no significant inorganic or nonvolatile organic residue was present in the material. In this case, wNV was assigned a value of 0 mg/g with u(wNV) of 0.28 mg/g. This was consistent with the reference value assigned for nonvolatile content in the digoxin material of 0.2 mg/g with an uncertainty of 0.06 mg/g. Mass Fraction of Nonvolatile Residue in Aldrin. The source material was technical grade aldrin that had been purified for comparison. The presence of chlorine precluded an exhaustive oxidative pyrolysis under TGA conditions, so only data from ICP-MS, elemental analysis, and 1H NMR studies were used to determine nonvolatile content. As these all indicated no significant nonvolatile materials, the combined nonvolatile content was assigned a value of 0 mg/g with the positive uncertainty increased by 30% to 0.4 mg/g to reflect the lack of supporting data from the TGA. Significant combined oligomeric organic impurities, at the level of 11 mg/g, were subsequently determined to be present in the material.9 The empirical formula of these unidentified impurities must be sufficiently close to that of aldrin such that no significant deviation from theoretical values for aldrin in the elemental analysis resulted, and being perchlorinated, they were also not detected in the 1H NMR spectrum. Subsequent investigations have revealed that the oligomeric impurity in this sample of aldrin can be detected and quantified by gel permeation chromatography. A technical capability for this analysis has been incorporated into the BIPM characterization protocols and give us the ability to control for impurities of this type when undertaking future purity assignments. Uses of 1H NMR Spectroscopy. Structural information obtained by NMR spectroscopy was used to verify the identity and amount of related structure impurity and residual organic solvent present in each material. In the case of aldrin, it also 3123



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Table 1. BIPM Mass Balance Content Assignments and Reference Values for Each Material mass fraction of component xj (mg/g); uxj (mg/g) comparison

combined related structure

BIPM values reference values

0.7 (0.2) 0.8 (0.3)


13.9 (0.55) 13.4 (0.6)


15.7 (0.6) 14.3 (0.6)

BIPM values reference values BIPM values reference values

8.9 (0.6)20 8.65 (0.13) 34.4 (0.65) 35.4 (0.42)

water

residual solvent

Theophylline (Pure) 0.2 (0.05) 0.2 (0.04) Theophylline (Spiked) 2.2 (0.25) 2.45 (0.25) Digoxin 1.1 (0.2) 1.3 (0.7) Estradiol 7.5 (0.44) 6.75 (0.44) Aldrin 0.5 (0.3) 0.5 (0.05)

nonvolatiles

main component

0 (−) 0 (−)

0 (−) 0 (−)

999.1 (0.2) 999.0 (0.3)

0 (−) 0 (−)

0 (−) 0 (−)

983.8 (0.65) 983.1 (1.5)

3.6 (0.2) 3.9 (0.2)

0 (0.3) 0.2 (0.06)

979.6 (0.65) 980.2 (1.0)

0 (0.1) 0.1 (0.06)

0 (0.3) 0.2 (0.12)

983.6 (0.9)20 984.3 (0.4)

2.5 (0.1) 2.3 (0.2)

0 (0.4) 11 (0.6)

963.1 (0.83) 950.8 (0.85)

In Figure 1, this data is displayed in the form of degree of equivalence (DOE) plots. The degree of equivalence (D) of each result x with standard uncertainty ux with respect to the reference value xR with standard uncertainty uR is given by a pair of terms:

provided an independent measure of the purity of the main component. For theophylline and aldrin, a quantification of the major related structure impurities was obtained by the integration of the unique signals for each impurity present in the 1H NMR spectrum using adjacent, well-resolved 13C satellite peaks of signals from the main component as an internal quantification standard. For digoxin and estradiol the number, structural complexity, and extensive overlap of the 1H NMR spectra of the impurities precluded obtaining useful quantification information on the impurity content. Inspection of the 1H NMR spectra was also used to confirm the residual solvent impurity assignments, independently verifying the conclusions of the more sensitive GC-MS studies. For aldrin a limited quantitative NMR (qNMR) analysis19 provided a direct assignment of the aldrin content of the material. The qNMR study was undertaken on aliquots from two units of the production batch using dimethylterephthalate (DMTP) as the internal quantification standard. The mass fraction of aldrin in the sample was assigned from the relative integral ratios of the unique signals for DMTP and aldrin with appropriate adjustment for the molar masses and relative absolute masses of DMTP and aldrin in each qNMR solution. In this case we were only able to perform a limited study and the relatively large variation in results obtained meant we were unable to detect with sufficient confidence that the mass balance approach had failed to detect the significant levels of oligomeric impurity in the aldrin. The qNMR results obtained by some participants in the comparison did however clearly reveal this difference and provides an alternative to size exclusion chromatography to check for significant levels of nonvolatile organics.9

D = (x − x R ) Ux = 2(ux 2 + uR 2)1/2

Where, Ux is the expanded uncertainty (k = 2) associated with D, with both expressed in units of milligrams per gram. The plots show that the BIPM estimate and the corresponding reference values agree within their stated uncertainties in every case, with the exception of the nonvolatile impurity content of the aldrin material. As noted above, in the case of estradiol the values reported in Table 1 and plotted for combined related structure impurity in Figure 1 are those obtained using methods that avoid related structure artifact formation and that were developed subsequent to the original comparison. The BIPM mass balance method performed extremely well in all respects when benchmarked against state of the art capabilities for organic purity assignment. Where biases have occurred, their cause has been identified and our approach revised accordingly. It delivers SI-traceable mass fraction purity assignments with small associated measurement uncertainties for a wide range of organic structural types and measurement challenges.

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EXPERIMENTAL SECTION Chemicals. The following reference materials for main component and actual or potential related structure impurities for each material characterized were used. For theophylline: theophylline, caffeine, theobromine, various dimethylxanthines and 1-, 3-, and 7-methylxanthine were obtained from Sigma-Aldrich (St. Quentin Fallavier, France). For digoxin: digoxin, digitoxin, digoxigenin, and digitoxigenin were obtained from Sigma-Aldrich (St. Quentin Fallavier, France). Desacetyllanatosid C, digoxigenin-tetra-digitoxoside, and lanatosid C were purchased from Carl Roth (Karlsruhe, Germany). Purpureaglycoside A, purpureaglycoside B, and ßacetyldigoxin were bought from EDQM (Strasbourg, France).

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PERFORMANCE OF THE BIPM MASS BALANCE PROCEDURE Table 1 summarizes the BIPM mass balance method assignments of the mass fraction of the impurity classes and the main component and compares them with the reference values assigned for each of the five compounds that have been the subject of purity assignment comparisons coordinated by the BIPM. The reported BIPM estradiol results are the corrected values obtained subsequent to the original comparison using methods that avoid artifact formation. 3124



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were processed at the BIPM using the Bruker TopSpin (version 2.0) software.

Gitoxin and gitoxigenin were obtained from ABCR (Karlsruhe, Germany) and Fluka (Schnelldorf, Germany), respectively For estradiol: Estradiol, estrone, and 4-methylestradiol were obtained from Sigma-Aldrich (St. Quentin Fallavier, France). 9,11-Dehydroestradiol was obtained from LGC Standards. For aldrin: Aldrin, dieldrin, and isodrin were obtained from Sigma-Aldrich (St. Quentin Fallavier, France). Dechlorane was obtained from Toronto Research Chemicals (Toronto, Canada). Solvents of HPLC gradient grade quality and reagents of analytical grade or better were purchased as required from Sigma-Aldrich (Lyon, France), Fluka (Lyon, France), and Merck. Preparation of Standard Solutions. A description of general procedures for preparation of standard solutions for LC and GC analysis and quantification, and the estimation of their associated uncertainties, is provided in a prior publication.13 Chromatography Systems and Conditions. The LC system consisted of an Agilent 1100 series micro vacuum degasser, binary pump, thermostatted standard autosampler, thermostatted column compartment, and diode array detector (DAD). An Applied Biosystems 4000 Qtrap hybrid tandem mass spectrometer was coupled to the LC system employing a Sciex TurboIonSpray (TIS) source and a Valco 10-position valve. A direct flow injection device from Harvard Apparatus was used for optimization by direct injection. LC methods with diode-array UV detection (UV) were developed for the determination separately of theophylline, digoxin, estradiol, and aldrin in the presence of a range of potential related structure impurities. Details of the individual methods used for the preparation of gravimetric solutions, LC conditions, and optimized ionization parameters are described in relevant comparison reports and publications for theophylline,6 digoxin,7,13 estradiol,8,17 and aldrin.9 The GC system consisted of an Agilent 7800 chromatograph connected to either an FID or to an Agilent 7850 Mass Selective detector. The chromatographic conditions for GC-MS for each material for detection of residual solvent and GC-MS and GC-FID for quantification of the related structure impurities in aldrin are described in the comparison reports. Karl Fischer Titration System and Conditions. Karl Fischer titration was performed using a Mettler DL 39 Karl Fischer Coulometric Titrator, operated by LabX Pro (version 2.0) software. Heated oven transfer determinations of water content were performed using a Mettler Stromboli Solid Sample Oven with the released water vapor transferred into the titrator cell through a stream of dried, high purity nitrogen gas. NIST Standard Reference Material 2890 (Water in 1-Octanol) was used to validate the operation and underpin the SI traceability of the results obtained by Karl Fischer titration. The specific titration conditions are described in the relevant comparison reports. Thermogravimetric System and Conditions. Thermogravimetric analysis was performed using a Perkin-Elmer Pyris 1 thermogravimetric analyzer with AccuPik autosampler. The TGA conditions used for each compound are described in the relevant comparison reports. NMR Systems and Conditions. 1H and 13C NMR spectra were obtained by the University of New South Wales Nuclear Magnetic Resonance facility (Sydney, Australia) as solutions in d6-DMSO (for theophylline and digoxin) and CDCl3 (for estradiol and aldrin). The data files obtained for each analysis

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CONCLUSION We describe a comprehensive implementation of the mass balance approach for purity assignment of organic compounds that uses complementary and independent techniques for detecting and quantifying the mass fraction of each of the four general impurity classes potentially present in a high purity organic compound. The related structure impurity content is quantified by external calibration using two independent high resolution chromatography techniques in tandem with selective, sensitive detectors and the assigned value is independently confirmed by 1H NMR data. The water content is quantified by Karl Fischer titration and confirmed by TGA and elemental analysis. The residual solvent content is quantified by GC-MS methods and confirmed from 1H NMR data. The nonvolatiles content is assessed by combining data from an ensemble of methods that give fit-for-purpose estimates of the inorganic, nonvolatile organic or combined nonvolatile impurities. Gel permeation/size exclusion techniques are also now used to control for oligomeric organic impurities. The traceability to the SI of each of the contributing impurity classes has been demonstrated such that we establish with suitable confidence that the assigned value for the mass fraction of the main component is also traceable to the SI. The standard uncertainties of the mass fraction of the main component obtained by this approach are in general significantly smaller than those achievable by direct assay methods. The applicability of the approach has been tested in a series of international comparisons of purity assignment. The results obtained were generally in excellent agreement with reference values established both for the purity of the main component and for the mass fraction of each impurity present in each material. The method has been refined and strengthened in the course of undertaking these assignments after reviewing our performance in each comparison in the light of the results obtained by all participants. The method provides a comprehensive and rigorous method for the SI-traceable assignment of the mass fraction purity of primary organic calibrators, appropriate in the majority of cases for their use as the highest order calibrator5 in a metrological traceability heirarchy.

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ASSOCIATED CONTENT

S Supporting Information *

Supporting material Figure 1 and Tables 1 and 2. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors wish to thank LGC Limited, the National Metrology Institute of Japan, the National Measurement Institute of China, and the Centro Nacional de Metrologia de Mexico for technical and logistical support to develop and undertake this work. Specific contributions to the development 3125



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of this approach by Chen Dazou (NIM), Toshihide Ihara (NMIJ), Yoshitaka Shimizu (NMIJ), Juan Guardado (CENAM), Charline Mesquida (BIPM), Steve Wood (LGC Limited), and Thierry Le Goff (LGC Limited) are also gratefully acknowledged.

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REFERENCES

(1) European Council Directive 96/23/EC of 29 April 1996 on measures to monitor substances and residues in live animals and animal products, 1996. (2) European Council Directive 98/79/EC of 27 October 1998 on in vitro diagnostic medical devices, 1998. (3) World Anti-Doping Code: International Standard for Laboratories, Version 7.0; World Anti-Doping Agency: Montreal, January, 2012. (4) ISO/IEC 17025. General requirements for the competence of testing and calibration laboratories; 2005 (5) ISO 17511. In vitro diagnostic medical devices-Metrological traceability of values assigned to calibrators and control materials; 2003. (6) Westwood, S.; Josephs, R. D.; Daireaux, A.; Wielgosz, R.; et al. Metrologia 2009, 46 (1A), 08019. (7) Westwood, S.; Josephs, R. D.; Choteau, T.; Daireaux, A.; Wielgosz, R.; et al. Metrologia 2011, 48 (1A), 08013. (8) Westwood, S.; Josephs, R. D.; Daireaux, A.; Wielgosz, R.; et al. Metrologia 2012, 49 (1A), 08009. (9) Westwood, S.; Josephs, R. D.; Daireaux, A.; Wielgosz, R.; et al. Metrologia 2012, 49 (1A), 08014. (10) Duewer, D.; et al. An Approach to the Metrologically Sound Traceable Assessment of the Chemical Purity of Organic Reference Materials; NIST Special Publication 1012; NIST: Gaithersburg, MD, 2004. (11) Vehovec, T.; Obreza, A. J. Chromatogr. A. 2010, 1217, 1549− 1556. (12) Ettre, L. S.; et al. Pure Appl. Chem. 1993, 65, 819−872. (13) Josephs, R. D.; Daireaux, A.; Westwood, S.; Wielgosz, R. I. J. Chromatogr. A 2010, 1217, 4535−4543. (14) JCGM. International Vocabulary of Metrology−Basic and General Concepts and Terms; 2012; JCGM 200:2012. (15) Eurachem Quantifying Uncertainty in Analytical Measurement; 2nd ed.; 2000; Annex A (16) Novakova, L.; Solich, P.; Matysova, L.; Sicha, J. Anal. Bioanal. Chem. 2004, 379, 781−787. (17) Josephs, R. D.; Westwood, S.; Daireaux, A.; Wielgosz, R. J. Chromatgr. A, submitted for publication. (18) Mettler Toledo Titrators DL32/DL39 Applications Brochure 32: Fundamentals of the Coulometric Karl Fischer Titration with Selected Applications; Mettler Toledo: Columbus, Ohio. (19) Jancke, H.; Malz, F.; Haesselbarth, W. Accredit. Qual. Assur. 2005, 10, 421−429. (20) Value assigned using data obtained from methods developed subsequent to the original comparison.

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Mass Balance Method for the SI Value Assignment of the Purity of

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