Role of chromatography in the development of Standard Reference Materials for organic analysis

Journal of Chromatography A, 1261 (2012) 3–22

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Review

Role of chromatography in the development of Standard Reference Materials for organic analysis夽 Stephen A. Wise ∗ , Karen W. Phinney, Lane C. Sander, Michele M. Schantz Analytical Chemistry Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA

a r t i c l e

i n f o

Article history: Available online 6 June 2012 Keywords: Standard Reference Material (SRM) Polycyclic aromatic hydrocarbons (PAHs) Gas chromatography–mass spectrometry (GC–MS) Liquid chromatography–mass spectrometry (LC–MS) Carotenoids Chromatographic selectivity

a b s t r a c t The certification of chemical constituents in natural-matrix Standard Reference Materials (SRMs) at the National Institute of Standards and Technology (NIST) can require the use of two or more independent analytical methods. The independence among the methods is generally achieved by taking advantage of differences in extraction, separation, and detection selectivity. This review describes the development of the independent analytical methods approach at NIST, and its implementation in the measurement of organic constituents such as contaminants in environmental materials, nutrients and marker compounds in food and dietary supplement matrices, and health diagnostic and nutritional assessment markers in human serum. The focus of this review is the important and critical role that separation science techniques play in achieving the necessary independence of the analytical steps in the measurement of trace-level organic constituents in natural matrix SRMs. Published by Elsevier B.V.

Contents 1.

2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. What are Standard Reference Materials? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Development of the independent methods approach for SRM value assignment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Modes of certification for chemical composition in SRMs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. Types of SRMs for chemical composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.5. Development of first SRM for trace organic constituents using multiple independent techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.6. Development of first-generation SRMs for trace organic composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Separation science selectivity to achieve method independence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. LC analysis for PAHs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Stationary phase/column selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Multidimensional LC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. GC–MS analysis of PAHs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Chromatographic and detection selectivity for PCBs and chlorinated pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Extraction approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Isolation and cleanup techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Quantification approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Current approach for determination of PAHs in environmental-matrix SRMs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.8. Multiple methods for other organic contaminants in environmental-matrix SRMs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

夽 Certain commercial equipment, instruments, or materials are identified in this report to specify adequately the experimental procedure. Such identification does not imply recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials or equipment identified are necessarily the best available for the purpose. ∗ Corresponding author. Tel.: +1 301 975 3112; fax: +1 301 926 8671. E-mail address: [email protected] (S.A. Wise). 0021-9673/$ – see front matter Published by Elsevier B.V. http://dx.doi.org/10.1016/j.chroma.2012.05.093

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3.

4.

Implementation of multiple methods approach for vitamins, carotenoids, and fatty acids in food and dietary supplements . . . . . . . . . . . . . . . . . . . . 3.1. Development of independent methods for vitamins and carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Water-soluble vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. Fat-soluble vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.3. Carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Development of independent methods for fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Development of independent methods for botanical dietary supplements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Ephedra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. Ginkgo biloba . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Development of independent methods for clinical health status markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Ever since the National Bureau of Standards (NBS), now the National Institute of Standards and Technology (NIST), was established in 1901, the production of standard materials for chemical composition has been a major activity. In 1905 the “Standard Samples” program was started with “standardized irons” in collaboration with the American Foundrymans Association. In 1906 at the request of the Association of American Steel Manufacturers, NBS initiated work on the certification of 17 types of steel. By 1951 there were 502 Standard Samples including 98 steels. In the mid-1960s the Standard Samples became Standard Reference Materials (SRMs), and today there are over 1300 different types of SRMs with approximately 1000 of the materials intended for chemical composition measurements. The early steel, iron, and ore samples (and most of the subsequent replacements) were generally certified for major chemical composition constituents. The first natural-matrix SRMs certified for trace elements and tracelevel organic constituents were developed in the early 1970s and early 1980s, respectively, for the determination of contaminants in environmental matrices and for clinical health status markers in human-serum matrices. 1.1. What are Standard Reference Materials? SRMs are certified reference materials (CRMs) issued by the National Institute of Standards and Technology. The current version of the International Vocabulary of Basic and General Terms in Metrology (VIM) [1] defines a reference material (RM) as: “Material, sufficiently homogeneous and stable with reference to specified properties, which has been established to be fit for its intended use in measurement or in examination of nominal properties.” The VIM defines a CRM as: “Reference material, accompanied by documentation issued by an authoritative body and providing one or more specified property values with associated uncertainties and traceabilities, using valid procedures.” For the SRMs discussed in this review, the “documentation” is provided in the form of a “Certificate of Analysis” and the “specified property values” are the chemical compositions typically reported as mass fractions. The Certificate of Analysis also generally provides a brief summary of the measurement methods used to determine the reported mass fraction values. 1.2. Development of the independent methods approach for SRM value assignment In 1971 NBS issued what many analytical chemists would consider as the first natural-matrix SRM for trace environmental chemical measurements, SRM 1571 Orchard Leaves, with certified concentrations of 19 major, minor, and trace element constituents.

12 13 13 14 15 16 16 17 18 19 20 21 21

The assignment of the certified values in this natural-matrix SRM was based on the approach of combining results from two or more independent and reliable analytical methods. The “independent method concept” for the certification of chemical composition of reference materials had been used since the early days at NBS, when W.F. Hillebrand, the chief chemist at NBS, stated that one criterion of a standard sample is that “its composition should have been determined by independent and reliable methods affording agreeing results” [2]. With the development of SRM 1571 Orchard Leaves, the independent methods concept as applied to naturalmatrix SRMs for trace element content was formalized. During the 1970s, additional natural-matrix SRMs for trace element content were developed including bovine liver, coal, fly ash, spinach, pine needles, water, river and estuarine sediment, air particulate matter, and oyster tissue. For the determination of the trace element concentrations in these natural matrices, the “two or more analytical techniques” approach was relatively straight forward, namely, the elements could be determined either directly in the solid matrix using non-destructive techniques such as neutron activation analysis and X-ray spectrometry, or the sample matrix could be dissolved (digested) and the total elemental content of the solution measured using multiple elemental analysis techniques (e.g., atomic absorption spectrometry, mass spectrometry, atomic emission spectrometry, and/or polarography). For the determination of trace elements in matrix SRMs, no separation methods (extraction or chromatography) were generally required. In 1991, Epstein [3] described the development of the independent methods concept as applied at NIST to certify elemental composition, primarily trace element content in environmental matrices. The objective of the multiple methods approach is to use methods that are as chemically independent as possible (i.e., different sources of bias) in all steps of the analytical measurement process. The results from these multiple techniques, if in agreement, are combined to determine the certified concentration (mass fraction) value of the analytes measured. The requirement for using two or more analytical techniques is based on the assumption that the agreement of the results from the independent methods minimizes the possibility of bias within the analytical methods. In the late 1970s NBS began to address the need for SRMs for the determination of trace organic constituents in complex environmental and clinical matrices. The implementation of the two or more analytical techniques criterion for the certification of individual organic constituents in natural environmental matrices presented a more challenging task compared to the implementation of the independent methods concept for trace element determinations. For the measurement of trace organic constituents, the compounds of interest must be removed (generally solvent extraction) from the solid matrix because the matrix cannot be dissolved, as is generally the case for trace element analysis, without destroying the organic constituents. The compounds of interest must then be isolated (a cleanup step) and/or enriched

S.A. Wise et al. / J. Chromatogr. A 1261 (2012) 3–22

Use of Independent Analytical Methods to Exploit Differences in Techniques

Extraction

Parameters

Soxhlet Ultrasonic Pressurized Fluid Supercritical Fluid Microwave-assisted Mechanically agitated

Solvent Temperature Pressure pH

Off-Line Approaches

Cleanup Isolation Enrichment

Liquid-Liquid Extraction Column Chromatography Liquid Chromatography Solid Phase Extraction (SPE) Solid Phase Microextraction (SPME)

Instrumental Approaches

Separation

Gas Chromatography (GC) Liquid Chromatography (LC) Ion Chromatography Electrophoresis Multidimensional Separation

GC

Detection

MS MS/MS FID ECD Flame photometric AED

Calibration

Quantification

External Standard Internal Standard Isotope Dilution Standard Addition

LC MS MS/MS Absorbance Fluorescence Electrochemical ELSD CAD

Model Linear Regression Slope/Intercept Zero Intercept Bracketed Calibration Exact Matching Nonlinear Calibration

Minimize the possibility of undetected bias in resulting certified concentrations Fig. 1. Use of independent analytical methods for certification of SRMs for organic analysis.

(concentrated) from the resulting extract. Finally the analysis step typically relies on the separation of the individual organic constituents using techniques such as gas chromatography (GC) or liquid chromatography (LC) generally with selective detection. In 1980 NBS issued the first natural-matrix SRM with certified concentrations for trace-level organic environmental contaminants, SRM 1580 Organics in Shale Oil, with certified values for a limited number of organic constituents. Hertz et al. [4] described the development of the multiple independent methods that were necessary for the certification of these organic constituents in the shale oil SRM, which required that all the steps in the analytical measurement process (e.g., extraction, isolation, chromatographic separation, and detection) be as independent as possible. The paper by Hertz et al. [4] provided a critical evaluation and comparison of the results obtained using the different methods, and thus established the validity of this approach for the certification of organic constituents in complex natural-matrix SRMs. Due to declining interest in alternate fuels in the early 1980s, SRM 1580 was not used widely, and it may have been considered by some to be a failure. However, the analytical approach defined as part of the certification of the shale oil SRM became the foundation for the subsequent development of over 30 natural environmental-matrix SRMs for organic contaminants. When applying the independent analytical methods approach to the determination of trace organic constituents, the goal is to exploit and maximize differences (ideally based on chemical principles) in each of the steps in the analytical process, i.e., extraction; cleanup, isolation, and/or enrichment; separation; detection; and quantification approach, as illustrated in Fig. 1. Within each step, multiple options may exist to utilize differences in “separation science” selectivity to achieve the analytical method independence.

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This review will describe the evolution of the multiple independent methods approach for organic constituents in natural-matrix materials through the first three decades of SRM development for organic environmental contaminants followed by the subsequent implementation of this approach to food and dietary supplement SRMs for organic nutrients and marker compounds, and to human tissue matrix SRMs (e.g., serum and urine) for health diagnostic and nutritional assessment markers. The focus of this review is the important and critical role that separation science techniques play in achieving the required independence of the analytical steps in the measurement of trace-level organic constituents in naturalmatrix SRMs. 1.3. Modes of certification for chemical composition in SRMs Historically, NIST has used three basic modes for SRM certification of chemical composition: (1) measurements using a “definitive or primary” method, i.e., a high-precision method for which sources of bias have been rigorously investigated, (2) measurements using two or more independent and reliable methods; and (3) measurements from a number of laboratories participating in a multi-laboratory comparison exercise, e.g., round robin or interlaboratory studies. In 2000 NIST reexamined the various approaches (modes) used for value assignment of chemical composition in SRMs and defined three terms (certified, reference, and information) to describe the values assigned to SRMs [5]. The three historical modes of certification mentioned above were expanded to seven modes, which are currently used at NIST for value assignment for chemical composition in natural-matrix SRMs [5]. These seven modes and the values resulting from each mode are summarized in Table 1. The basic principles for value assignment remain unchanged from the early days at NBS; however, these modes now provide a well-defined link between the approach used for value assignment and the level of confidence that NIST has in the accuracy of the assigned value (i.e., certified values have the highest level of confidence). For natural-matrix SRMs the predominant mode for value assignment is the use of two or more independent analytical methods (mode 2). The use of definitive methods (mode 1) for value assignment has been used primarily for measurements of analytes of clinical importance as health-status markers as will be described later. 1.4. Types of SRMs for chemical composition SRMs for chemical composition are used primarily for the following purposes: (1) to calibrate the measurement system; (2) to validate the accuracy, reliability, and reproducibility of a new analytical method; (3) to provide quality control of routine measurements by analyzing a reference material at appropriate, regular time intervals (control charting); and (4) as tools to provide metrological traceability to international standards. Two types of SRMs for chemical composition are available from NIST with different intended uses: (1) solutions containing a number of elements or compounds and (2) natural-matrix materials. The solution SRMs intended for organic analysis are useful for validating and calibrating the chromatographic separation/detection step (determining the detector response for a given mass of element/compound) in the measurement process. The natural-matrix SRMs for organic analysis, which are often similar to the actual samples analyzed, are used to evaluate/validate the complete analytical procedure including solvent extraction, cleanup of the extract, isolation/enrichment of the compounds of interest, and the final chromatographic separation, detection, and quantification. Although chromatography plays a role in the development and value assignment of both solution and matrix SRMs, the focus of this review will be on the

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Table 1 Modes used at NIST for the assignment of chemical composition values to reference materials. NIST certified value 1 2 3 4 5 6

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1.5. Development of first SRM for trace organic constituents using multiple independent techniques For the shale oil SRM, the development and implementation of the multiple independent techniques approach focused on three steps in the analysis process: (1) isolation, (2) separation, and (3) detection with all three steps involving the use of chromatography. In the late 1970s the analysis of petroleum or alternate fuel materials typically focused on isolation of compound classes using an acid–base extraction scheme to separate neutral, acid, and base fractions or low-resolution adsorption or ion-exchange chromatography to isolate various chemical class fractions for further characterization. The compounds of primary interest for the shale oil were polycyclic aromatic hydrocarbons (PAHs) (neutral fraction) and phenols (acid fraction). A critical element in the establishment of this independent methods approach for the analysis of the shale oil was the development of a normal-phase LC method using a polar, chemically bonded stationary phase (aminopropylsilane) to isolate the PAHs (or other compounds of interest) in fractions that were subsequently analyzed by using GC and/or LC. This LC fractionation approach provided the second cleanup approach to complement the classical acid–base extraction. For the analysis step, LC and GC provided independence in the separation process based on very different compound/chromatographic stationary phase interactions. For the GC analyses the universal flame ionization detector (FID) and a highly selective detector (mass spectrometry, MS) were used, and for the LC analyses selective ultraviolet (UV) absorbance and fluorescence (FL) detection were used. In addition to focusing on the independence of the extraction/isolation, separation, and detection, different approaches to quantification were investigated including external standards and internal standards (added prior to or after extraction or LC fractionation) to determine detector response factors. For the internal

NIST information value

Y

Certification at NIST using a single primary method with confirmation by other method(s) Certification at NIST using two independent critically evaluated methods Certification/value-assignment using one method at NIST and different methods by outside collaborating laboratories Value-assignment based on a method-specific protocol Value-assignment based on measurements by two or more laboratories using different methods in collaboration with NIST Value-assignment based on NIST measurements using a single method or measurements by an outside collaborating laboratory using a single method Value-assignment based on selected data from interlaboratory studies

natural-matrix SRMs for the determination of trace organic constituents.

NIST reference value

Y

Y

Y

Y Y Y

Y Y

Y

Y

Y

Y

standard approach, different compounds were often used as the internal standards for the same analyte measured by different techniques. The results of the analyses of the shale oil SRM using the multiple analytical techniques are summarized in Table 2 for four PAHs and two phenols, and the procedures are described in detail in Hertz et al. [4]. Following the LC extraction as the isolation step, the individual PAHs and phenols were determined by using LC with fluorescence (for the PAHs) and UV absorbance (for the phenols), GC–FID, and GC–MS. GC–FID and GC–MS analyses were performed on the neutral (PAHs) and acid (phenols) fractions isolated from the shale oil through an acid–base extraction scheme. Both the LC extraction and the acid–base extraction were successful in providing a fraction suitable for GC analysis with the universal FID. A third independent approach with no extraction or cleanup was possible using the highly selective MS detection, i.e., direct injection of the complex shale sample into the GC for determination of two PAHs. This direct analysis approach provided the necessary method independence for the SRM certification but was not practical for routine analysis because of the rapid degradation of the GC columns. Considering the state-of-the-art of trace organic analysis in 1980, the agreement of the results from the various techniques (shown in Table 2) was very good. 1.6. Development of first-generation SRMs for trace organic composition Following the development of the shale oil SRM in 1980, six other natural-matrix SRMs were issued during the next decade with certified concentrations for a limited number of PAHs including petroleum crude oil [6], air and diesel particulate matter [7,8], coal tar extract [9], marine sediment [10,11], and mussel tissue [12]. The focus on the measurement of PAHs in these first-generation SRMs was fortunate from the perspective of analytical methods development. PAHs represented an important group of ubiquitous, combustion-related contaminants that appear in environmental samples as a complex mixture containing numerous isomeric

Table 2 Results of the analysis of SRM 1580 Organics in Shale Oil using multiple analytical techniques. Results are reported as mass fraction (mg/kg). Uncertainties are 95% confidence level. LC fractionation, quantification by

Acid/base extraction, quantification by

No extraction, quantification by

Compound

LC

GC–MS

GC–MS

GC–MS

Pyrene Fluoranthene Benzo[a]pyrene Benzo[e]pyrene Phenol o-Cresol

108 ± 16 53 ± 6 21 ± 3 – 383 ± 50 330 ± 34

– – 24 ± 2 22 ± 5 334 ± 63 322 ± 45

104 ± 8 58 ± 5 – – – –

Adapted from Ref. [4].

GC 101 ± 4 55 ± 6 – – 387 ± 26 334 ± 86

102 ± 9 62 ± 5 21 ± 5 20 ± 6 416 ± 28 350 ± 16

GC 94 ± 10 75 ± 5 – – –

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structures. The presence of numerous isomers in the complex mixtures, including methyl- and other alkyl-substituted isomers, provided significant challenges and opportunities in the development of the multiple independent methods. The shale oil, petroleum crude oil, and coal tar SRMs represented complex mixtures in a liquid form. The air and diesel particulate matter, sediment, and mussel tissue SRMs represented solid matrices, which required a solvent extraction step to remove the constituents of interest from the solid matrix, thereby introducing another step requiring independent approaches. During the first decade of development of these environmental SRMs, the precision and accuracy of the analytical methods improved for PAH measurements, and the multiple analytical methods approach for assigning certified values for PAHs was firmly established. Analytical method improvements included the use of: (1) perdeuterated PAHs as internal standards for quantification in both GC–MS and reversed-phase LC–FL analyses [6], (2) more selective C18 stationary phases for the reversed-phase LC analysis [7,13,14], (3) fluorescence spectrometers with excitation and emission wavelength programming to increase the selectivity of the detection in the LC analyses [7,9], and (4) a multidimensional LC procedure to isolate isomeric PAHs by normal-phase LC prior to analysis by using reversed-phase LC for separation of the PAH isomers [15,16]. All these improvements expanded on the concept of chromatographic and detection selectivity as a means to provide independence of the measurements. At the beginning of the second decade of developing SRMs for the determination of PAHs, all of the procedures and concepts were in place to significantly expand the number of PAHs with certified values using the multiple independent methods approach. For the first generation of organic environmental-matrix SRMs, certified concentrations were typically provided for only 5–10 PAHs [17]. In addition to the certified values for these matrix SRMs, an additional 5–25 PAHs were typically reported as noncertified (now denoted as reference) values, primarily because of the lack of measurements by the requisite second independent method. Two significant challenges existed for increasing the number of PAHs with certified concentrations in natural-matrix SRMs. For the certification of PAHs in natural-matrix SRMs during the 1980s, the two primary analytical techniques were GC–MS and LC–FL. GC–MS using a 5% phenylmethylpolysiloxane phase was the common procedure for the measurement of PAHs at the time (and still is in many laboratories today) based in part on the landmark GC open tubular column and stationary phase research of Lee et al. [18–20] and the development of retention indices for PAHs using this stationary phase [21]. GC–MS analysis of complex environmental PAH mixtures typically provided accurate quantitative results for 20–25 major parent PAHs based on separation selectivity for PAH isomers and the availability of authentic reference compounds of known, reliable purity. Reversed-phase LC–FL analysis of complex environmental PAH mixtures, however, typically provides results for only 10–12 PAHs (see Fig. 2) because of the lower chromatographic resolution and detector selectivity compared to GC–MS, thereby limiting the number of PAHs measured by two independent techniques as required to classify the assigned value as a NIST certified value. The understanding and manipulation of chromatographic selectivity in both LC and GC became a critical part of this challenge.

2. Separation science selectivity to achieve method independence 2.1. LC analysis for PAHs 2.1.1. Stationary phase/column selection For the LC determination of PAHs in complex mixtures, a critical factor is the selection of the appropriate stationary phase/column

7

to achieve the required separation. In the early 1980s reversedphase LC on C18 stationary phases was demonstrated to provide excellent separations for the major PAHs of interest (e.g., the U.S. Environmental Protection Agency priority pollutant list of PAHs). However, it also became apparent that not all C18 columns provided the same selectivity (i.e., relative separation) for PAHs [13,22,23]. A key finding in these investigations was that PAH separations were greatly influenced by the synthesis method used to prepare the C18 stationary phase. The majority of the C18 stationary phases at that time (and even today) are prepared by reaction of monofunctional silanes with silica to form “monomeric” bond linkages, whereas a “polymeric” phase is formed by using trifunctional silanes in the presence of water to form silane polymers on the silica surface [24]. Although the polymeric C18 phase is conceptually not well defined compared to the monomeric C18 phase, the chromatographic selectivity of the polymeric C18 phase for reversed-phase LC separation of PAHs is unique and exceptional. In a series of investigations by Sander and Wise [14,24–26], a fundamental understanding of the stationary phase characteristics responsible for the improved LC separations of PAHs on polymeric C18 were identified. The results of these studies of LC selectivity for PAHs have been summarized in several review papers [27–30]. Today, many LC column manufacturers identify their polymeric C18 columns specifically for PAH analyses. The term “shape selectivity” is commonly used to describe a chromatographic quality exhibited by a stationary phase that is defined by its ability to separate isomers or related compounds based primarily on solute shape, rather than other physical or chemical properties of the solutes. Polymeric C18 columns exhibit a high degree of shape selectivity; however, other columns and operating conditions also exhibit shape recognition. In general, ordered systems provide a higher degree of shape recognition than disordered systems [28], e.g., longer alkyl chain length stationary phase columns (e.g., C30 phase), use of subambient separation conditions, and liquid crystalline stationary phases (for GC) (see discussion below). 2.1.2. Multidimensional LC As shown in Fig. 2 (lower chromatogram), even using wavelength-programmed fluorescence detection to achieve selectivity and sensitivity, only about 10 major parent PAHs are reliably quantifiable. To increase the number of PAHs that could be quantified by LC, a multidimensional LC procedure was developed to isolate and enrich specific isomeric PAHs that are not easily measured using LC–FL in the total PAH fraction. This multidimensional LC procedure, which has been described in detail previously [7,9,16,31], consists of a normal-phase LC separation of the extract on an aminopropylsilane stationary phase followed by a reversedphase LC separation on a C18 stationary phase. The normal-phase LC step provides a separation of PAHs based on the number of aromatic carbon atoms in the PAH (approximately the number of aromatic rings), thereby providing fractions containing only isomeric PAHs and alkyl-substituted isomers [7,8,16]. These isomeric PAH fractions are then analyzed by reversed-phase LC–FL to separate and quantify the various isomers. This multidimensional approach has been described in detail in several papers reporting SRM certification measurements [9,11,32,33] and in a review paper [31] and book chapters [34–36]. An example of the multidimensional LC approach is shown in Fig. 3, which illustrates the normal-phase LC separation of a coal tar extract (SRM 1597) and the subsequent reversed-phase LC–FL analysis of two PAH isomer fractions, the first containing isomeric PAHs of molecular mass 278 Da (five-ring cata-condensed PAHs) (upper left chromatogram) and the second containing isomeric PAHs of molecular mass 302 Da (six-ring peri-condensed PAHs) (upper right chromatogram) [32]. Quantification of these isomeric PAHs of molecular mass 278 Da and 302 Da would be impossible using

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SRM 1941a Organics in Marine Sediment Four Ring Isomer Fraction

Six Ring Isomer Fraction Anthanthrene

Benzo[a]anthracene-d12

Benzo[a]anthracene

1 = 252/352 2 = 285/385 3 = 263/358

Indeno[1,2,3-cd]pyrene

1 = 380/405 2 = 300/500 3 = 310/430 Chrysene

Benzo[ghi]perylene Benzo[ghi]perylene-d12

Triphenylene Triphenylene-d12

1

3

2 5

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1 = 2 = 3 = 4 = 5 = 6 = 7 = 8 = 9 = 10 =

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8

9 40

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Fig. 2. LC–FL analysis of total PAH fraction (lower chromatogram) from SRM 1941a Organics in Marine Sediment with LC–FL analysis of four-ring (upper left chromatogram) and 6-ring PAH fractions (upper right chromatogram). Peak numbers 1, 3, 6, 11, and 14 are perdueterated PAH analogues of the peaks that they precede and are used as internal standards for quantification. Adapted from [11]; see reference for details of chromatographic conditions.

LC–FL analysis of the total complex PAH mixture. The use of the multidimensional LC approach to improve the measurement of the selected PAHs is illustrated in Fig. 2. Following the normalphase LC separation, reversed-phase LC–FL analyses of the four-ring cata-condensed PAH isomer fraction (upper left chromatogram) and of the six-ring peri-condensed PAH isomer fraction (upper right chromatogram) demonstrate the improvement compared with the reversed-phase LC–FL analysis of the total PAH fraction (Fig. 2 lower chromatogram). For the four-ring isomers, triphenylene would be difficult to quantify in the total PAH fraction, but it is easily measured after enrichment and isolation using normalphase LC. Similarly benzo[ghi]perylene, indeno[1,2,3-cd]pyrene, and anthanthrene are difficult to determine in the total PAH fraction due to the low concentrations and complexity of the sample. The multidimensional LC approach requires the addition of internal standards for each isomer fraction isolated (e.g., perdueterated triphenylene in Fig. 2 upper left chromatogram and perdeuterated benzo[ghi]perylene in Fig. 2 upper right chromatogram).

2.2. GC–MS analysis of PAHs The second challenge to increasing the number of certified PAH concentration values was the lack of separation of several important isomers on the 5% phenylmethylpolysiloxane phase commonly used in the GC–MS determination of PAHs. For example, the following isomeric PAHs pairs are not completely resolved on a 5% phenyl phase: triphenylene and chrysene; benzo[b]fluoranthene and benzo[j]fluoranthene; dibenz[a,c]anthracene and dibenz[a,h]anthracene. Reversed-phase LC on a polymeric C18 column provides baseline separation of these isomer pairs, but the separation by GC–MS on the 5% phenyl phase was the limiting factor to assigning certified values for these PAHs. The initial solution to this problem was the use of a smectic liquid crystalline stationary phase developed by Lee et al. [37–39], which provided shape-selective separations of PAH isomers similar to that of the polymeric C18 phase in LC. (A paper by Wise et al. [40] investigated the similarities in shape selectivity for PAHs using GC and LC stationary phases.) The smectic liquid crystalline phase provided

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SRM 1597 Coal Tar Excitation/Emission (nm)

Excitation/Emission (nm) 0 1 2 3 4

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x40

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60

80

100

Time (min) Fig. 3. Multidimensional LC approach for the determination of PAHs. Lower chromatogram: normal-phase LC analysis of SRM 1597 Complex Mixture of PAHs from Coal Tar. Upper left chromatogram – reversed-phase LC–FL analysis of isomeric five-ring PAH (278 Da molecular mass) fraction. Upper right chromatogram – reversed-phase LC–FL analysis of isomeric six-ring PAH (278 Da molecular mass) fraction. Adapted from Refs. [43,32]; see references for details of chromatographic conditions.

SRM 1650b Diesel Particulate Matter MW 228 isomers

MW 252 isomers benzo[b, j, + k]fluoranthene

triphenylene

nonpolar (DB-XLB)

50% phenyl methylpolysiloxane (DB-17)

dimethyl 50% liquid crystal polysiloxane

chrysene

benzo[a]fluoranthene

benz[a] anthracene

benzo[e]pyrene

benzo[c] phenanthrene

benzo[a]pyrene

benz[a] anthracene

benzo[b]fluoranthene

chrysene + triphenylene

benzo[k]fluoranthene benzo[a] fluoranthene

benzo[c] phenanthrene

benzo[a] fluoranthene

benz[a]anthracene triphenylene benzo[c]phenanthrene

chrysene

benzo[e]pyrene benzo[a]pyrene (perylene)

benzo[b]fluoranthene benzo[k]fluoranthene benzo[e]pyrene benzo[a]pyrene

benzo[j + a] fluoranthene

(perylene)

Fig. 4. GC–MS analysis of SRM 1650b Diesel Particulate Matter using three different stationary phases for the determination of PAHs of molecular mass 228 Da and 252 Da.

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GC separations of the critical PAH pairs mentioned above; however, the widespread use of the smectic phase was hampered by variations in selectivity among different columns (similar variations were also observed with early polymeric C18 LC stationary phases and these differences in column performance were the motivation for several investigations of stationary phase differences [14,22]) and limited temperature range. In spite of these limitations, GC–MS analysis using the smectic liquid crystalline phase provided the necessary second independent method for assigning certified values for PAHs in several early environmental-matrix SRMs [9,11,41]. In the early 2000s an improved liquid crystalline phase consisting of 50% dimethylpolysiloxane and 50% liquid crystalline polysiloxane became available commercially with a greater operating temperature range than the previous smectic phase [42]. A third GC stationary phase, 50% phenyl methylpolysiloxane, came into use in the late 1990s and provided separations of two of the critical PAH isomer pairs (benzo[b]fluoranthene and benzo[j]fluoranthene and dibenz[a,c]anthracene and dibenz[a,h]anthracene) as well as separations of additional isomers of these two isomer pairs. The GC separations of the isomeric PAHs of molecular mass 228, and 252 Da on three stationary phases are illustrated in Fig. 4. A relatively nonpolar, extra low bleed, proprietary phase (DB-XLB, Agilent, Wilmington, DE), resolves chrysene and triphenylene sufficiently to provide reliable quantitative results (see Fig. 4). Thus, the GC–MS analyses on the four different stationary phases (5% phenyl, DBXLB, 50% phenyl, liquid crystalline) with different selectivity for PAH separations complement the two LC procedures (i.e., total PAH fraction analysis and the multidimensional analysis) to potentially provide results from multiple independent techniques for a significant number of PAHs. An example of the use of chromatographic selectivity for value assignment of SRMs is the determination of PAH isomers of molecular mass 302 Da, i.e., the dibenzopyrenes and dibenzofluoranthene isomers in coal tar SRM. The dibenzopyrenes/fluoranthenes represent a large group of PAH isomers (33 possible isomers) that have received attention because of their carcinogenic potential. The separation selectivity of the polymeric C18 phase compared to the monomeric C18 phase is dramatically illustrated in Fig. 5 for the separation of dibenzopyrene/fluoranthene standards [43]. The differences in selectivity for the GC separation of these same isomers in the coal tar SRM on three different stationary phases are illustrated in Fig. 6 [44]. The comparison of the selectivity of the 50% liquid crystalline compared with the 5% phenyl phase in GC is similar to the comparison of the polymeric versus monomeric C18 phases in LC. Both the 50% phenyl phase and the 50% liquid crystalline phase provide excellent and somewhat different selectivity for the separation of the 302 Da molecular mass isomers as shown in Fig. 6. To achieve the independent methods for value assignment of these PAH isomers, three methods were implemented: (1) a multidimensional LC procedure [32], (2) GC–MS on a 50% phenyl phase, and (3) GC–MS on the 50% liquid crystalline phase. The multidimensional LC approach is illustrated in Fig. 3 and the GC–MS analyses on the 50% phenyl and 50% liquid crystalline phase are shown in Fig. 6B and C, respectively. The results from these three approaches were reported for the re-analysis and recertification of the coal tar SRM, which was issued as SRM 1597a [45]. 2.3. Chromatographic and detection selectivity for PCBs and chlorinated pesticides As described above, the focus of the first environmental-matrix SRMs was the determination of PAHs; however, SRMs for the determination of polychlorinated biphenyls (PCBs) and chlorinated pesticides were soon to follow, often measured in the same SRM matrix as the PAHs. The PCBs represented another important class of environmental contaminants containing numerous isomeric

3,4,6,8

7 12 9, 10

5

2 11 1

7

1

3

8 5

4 6 2

9 11 12 10

0

5

10

15

20

25

30

35

minutes Fig. 5. LC separation of PAH isomers of molecular mass 302 Da using a monomeric C18 vs. a polymeric C18 phase. Adapted from Ref. [43], see reference for chromatographic conditions.

structures in complex mixtures, which provided the opportunity to investigate and use stationary phase selectivity in achieving multiple independent methods for determination. The evolution of the multiple analytical methods approach for the determination of PCBs and chlorinated pesticides has been described in detail in several papers [46–49] including a review paper [50]. 2.4. Extraction approaches For trace organic analysis of solid samples (e.g., particulate matter, sediment, tissue), the first step in the analytical process is an extraction step, with solvent extraction generally the most common approach. In the development of the approach for certification of organic constituents in solid natural-matrix SRMs, two significant and related challenges regarding extraction were addressed: (1) determining whether all of the analyte of interest has been removed from the solid matrix, and (2) achieving selectivity in the extraction step. When the first particulate matter SRM (SRM 1649 Urban Dust) was issued in 1981 with certified values for selected PAHs [7], the question of whether all the PAHs were extracted from the particulate matter was heavily debated among the analysts at NIST. Previous NIST experience for certification of particulate matter SRMs was based on measurements of trace elemental composition, and therefore the concept of assigning a value for the total content of an element after dissolution of the particulate matrix was a reasonable approach. However, for the determination of PAHs or other organic contaminants in air particulate matter, it is not

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5% phenyl MPS (60 m)

A 62

64

66

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of PAHs, PCBs, and chlorinated pesticides from environmentalmatrix SRMs. The independence in the extraction method was achieved (to some extent) by using different solvents (e.g., hexane, dichloromethane, acetone, and methanol). By the late 1990s, supercritical fluid extraction (SFE) and pressurized fluid extraction (PFE), also known as pressurized liquid extraction (PLE), were being evaluated as alternatives to Soxhlet extraction. SFE conditions were found to be dependent on both compound and matrix [51–55]. In many of the natural-matrix SRMs, more than one class of compounds is often certified. For example in sediment and mussel tissue SRMs, PAHs, PCBs, chlorinated pesticides, and polybrominated diphenyl ethers (PBDEs) are often the targeted analytes, thus making SFE unsuitable as an alternative to Soxhlet extraction for certification of all of these contaminants in a natural-matrix SRM. PFE, based on using liquid solvents at elevated temperatures and pressures, was first introduced by Richter et al. [56] and later evaluated for the extraction of environmental-matrix SRMs by Schantz et al. [57]. The evaluation by Schantz et al. [57] demonstrated that PFE was a suitable alternative to Soxhlet extraction for removal of PAHs, PCB congeners, and chlorinated pesticides from environmental-matrix SRMs. However, higher extraction efficiencies were found for some of the higher molecular mass PAHs in the diesel particulate matter SRM using PFE compared to Soxhlet extraction (both using dichloromethane) [57]. Additional investigations using PFE for the extraction of PAHs from diesel and air particulate materials have suggested that using higher temperatures (>100 ◦ C) for the PFE conditions leads to higher extraction efficiencies for selected PAHs, particularly higher molecular mass PAHs and those with a linear structure [58–61]. The choice of solvent, pressure, and other PFE parameters does not have a significant effect on the extraction efficiency [61].

24 25

120

11

140

2.5. Isolation and cleanup techniques

160

Time (min) Fig. 6. GC–MS of PAH isomers of molecular mass 302 Da on three different stationary phases: (A) 5% phenyl phase, (B) 50% phenyl phase, and (C) 50% liquid crystalline phase. For identification of PAHs isomers and chromatographic conditions, see Ref. [44].

a viable approach to dissolve the particulate matrix and leave the PAHs intact as in the case of elemental measurements. Removal of the PAHs from the particulate matter using solvent extraction was necessary, and different extraction methods were investigated extensively (Soxhlet and ultrasonic extraction were the common methods at the time) using different solvents (hexane, cyclohexane, methanol, dichloromethane) in an attempt to demonstrate that all, or at least as much as possible of the PAHs, were removed. The extraction methods used to provide the certified values were described in detail on the SRM Certificate of Analysis, with the intent that if significant advances in extraction techniques in the future provided higher recoveries of PAHs from the SRM matrix, the certified values would be updated to reflect these improvements. In the past three decades, additional extraction techniques have been developed to provide more possibilities for independence and possibly different selectivity in the extraction step (see discussion below). The question of whether all of the PAHs are being extracted remains unanswered. Regarding extraction of PAHs from air and diesel particulate matter, are the PAHs adsorbed on the particle surfaces or are some PAHs incorporated into the particles during formation and therefore are not accessible to the extraction solvent? Is the measurement of PAHs inside a particle relevant? All of these questions are still debated. Until the late 1990s, Soxhlet extraction using a variety of solvents was the only method employed at NIST for the extraction

In addition to using different extraction methods, there is an attempt to have independent isolation and cleanup procedures for the analytical methods used in the certification of naturalmatrix SRMs. For isolation of contaminants from sediments and particulate matter samples, the approach typically involves using different SPE columns with different solvent mixtures for isolation of the analytes of interest or semi-preparative LC methods to isolate specific fractions. For biological samples, this first isolation step is typically used to separate the large quantities of lipids from the trace-level analytes of interest. The two techniques commonly used for this step are size-exclusion chromatography (SEC) and sulfuric acid treatment. Unfortunately, the sulfuric acid treatment can destroy some of the pesticides of interest, particularly dieldrin. After the lipids are discarded, different SPE columns with different solvent mixtures or semi-preparative LC are used to further isolate the analytes of interest. The different analytical methods used for the certification of PCB congeners and chlorinated pesticides in a biological matrix, SRM 1946 Lake Superior Fish Tissue, are described in detail by Poster et al. [46]. In this example, seven sets of measurement results were used for assigning the certified values including six NIST methods that illustrated the use of different extraction methods (Soxhlet and PFE using either dichloromethane or a hexane/acetone mixture), different isolation and cleanup methods (SEC, semi-preparative LC, and SPE), and the different separation and detection options [46]. 2.6. Quantification approaches The current quantification approach used for environmental analyses of natural-matrix SRMs is to add isotopically labeled internal standards, typically perdeuterated analogues for the PAHs and carbon-13 labeled analogues for the PCBs and pesticides, prior to

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extraction step. The same nominal quantity is added to the samples and to the calibration solutions, and then the calibration solutions are processed through the entire analytical procedure (extraction, isolation, clean-up, and final analysis step) in the same manner as the samples. Using this approach, the calibration solution acts both as a response and recovery solution, assuming no matrix effects. The use of internal standards reduces the need for quantitative transfers during the sample preparation steps and reduces biases from sample processing losses. The current certification methods for PAHs typically use at least one perdeuterated PAH for each molecular mass group of PAHs being quantified, as well as at least one carbon-13 labeled PCB for each congener molecular mass group. For the chlorinated pesticides, typically four or more carbon-13 or deuterated pesticides are used. Because there is not a corresponding isotopically-labeled compound for each analyte quantified, this approach has not been denoted as an isotope dilution method as is the case for methods for some clinical analytes (see discussion below). The use of carbon-13 labeled PAHs versus perdeuterated PAHs as the internal standards was investigated for the determination of PAHs in several environmental-matrix SRMs. As shown in Fig. 7A, the choice of perdeuterated PAHs or carbon-13 labeled PAHs as internal standards has no effect on the accuracy or precision of the PAH quantification for an air particulate matter material (SRM 1649b). However, it is important to have a labeled internal standard for each molecular mass PAH group that is being quantified as illustrated in Fig. 7B for the analysis of diesel particulate matter (SRM 1650b). In this study the results for the quantification of coronene (molecular mass 300) and dibenzofluoranthene and dibenzopyrene (molecular mass 302) resulted in higher mass fractions when using perdeuterated 302 molecular mass PAH as the internal standard compared to when using a labeled molecular mass 278 PAH as the internal standard. Using mussel tissue (SRM 1974b) as an example (see Fig. 7C), no differences in the accuracy or precision of the measurements for PCB congeners were observed when using a PCB congener that is not present in the sample compared to using a carbon-13 labeled PCB congener as internal standards or when using deuterated pesticides compared to carbon-13 labeled pesticides as the internal standards (Fig. 7D). 2.7. Current approach for determination of PAHs in environmental-matrix SRMs The current options for achieving independent analytical methods for the determination of PAHs in environmental matrix SRMs are summarized in Fig. 8 for each step in the analytical measurement process. Examples of the analytical approach used for assignment of mass fraction values for PAHs in natural-matrix SRMs are provided in several papers describing the certification of SRM 1944 New York/New Jersey Waterway Sediment and SRM 1941b Organics in Marine Sediment [33], SRM 1650 Diesel Particulate Matter [62], SRM 1649a Urban Dust [63], and SRM 2585 Organic Contaminants in House Dust [64]. In recent assignment of values for PAHs, LC–FL has been phased out of the analytical scheme due in part to the fact that the current GC phases provide the selectivity necessary for all of the critical isomer separations, and this approach is less labor intensive that the LC procedures. 2.8. Multiple methods for other organic contaminants in environmental-matrix SRMs In addition to PAHs, PCB congeners, and chlorinated pesticides, other groups of environmental contaminants are currently determined in natural-matrix SRMs including nitro-substituted PAHs (nitro-PAHs) [65,66], PBDE congeners, toxaphene congeners [67], and perfluorinated compounds (PFCs) [68]. Bamford et al. [66]

quantified nitro-PAHs in air particulate and diesel particulate SRMs using an amino/cyano semi-preparative LC column for isolation of a PAH fraction, mononitro-PAH fraction, and dinitro-PAH fraction prior to GC-negative chemical ionization (NICI) MS analysis. The authors demonstrated the need for using the LC isolation step to accurately identify and quantify the nitrofluoranthene and nitropyrene isomers, particularly in air particulate samples, and the need to use both a 50% phenyl methylpolysiloxane column and a 5% phenyl methylpolysiloxane column because of the differences in separation selectivity. The 50% phenyl phase provides baseline resolution of the 2-nitro- and 3-nitrofluoranthene isomers, which are not resolved on the 5% phenyl phase. In contrast, the 5% phenyl phase separates the 1-nitrobenzo[e]pyrene from the 6-nitrobenzo[a]pyrene, where as these two isomers are not separated using the 50% phenyl phase. Recently, the use of the 50% liquid crystalline polysiloxane phase has been added for certification measurements for nitro-PAHs in the air and diesel particulate SRMs. Similar to the separations of PAHs, the liquid crystalline phase provides shape-selective separations with the nitro-PAH isomers retention based on length-to-breadth ratio (solute shape) in addition to vapor pressure, thus resulting in a large change in the elution order in some cases when compared with conventional phases. Certified values for PBDE congeners are typically assigned using results from GC–MS analyses with electron impact (EI) and NICI sources and using a relatively nonpolar column and on-column injection. The use of short columns (30 m or less) and on-column injection is particularly important for quantifying the fully brominated PBDE 209 congener, which has been shown to debrominate in the injection port or if retained too long in the column [69]. Swarthout et al. [70] used GC coupled with inductively coupled plasma mass spectrometry (GC–ICP-MS) for the determination of PBDE congeners in sediment and mussel tissue SRMs (SRM 1941b and SRM 2977, respectively). They quantified 13 PBDE congeners in the two SRMs, including PBDE 209 in the sediment and found that the results were comparable (within 13%) to those previously reported [70].

3. Implementation of multiple methods approach for vitamins, carotenoids, and fatty acids in food and dietary supplements Micronutrients are a family of compounds and minerals that are essential to human health. With the exception of vitamin D and pantothenic acid, vitamins are not produced in the body and must be obtained through the diet, either from food or through supplementation. Vitamins serve a catalytic role in metabolism, and vitamin deficiencies result in illnesses that are usually reversible when sufficient levels are restored. The measurement of vitamin levels in foods and supplements permits assessments concerning adequacy of the diet, and health status can be assessed through determination of vitamin metabolites in the blood. Vitamins are usually categorized as fat-soluble or water-soluble vitamins based on their polarity and location within the sample matrices. Naturally occurring vitamins are conjugated to proteins or otherwise bound to matrix constituents, and extraction methods must include provisions for release from the matrix. In processed foods, vitamin levels are commonly fortified through the addition of synthetic vitamins, and recovery of these vitamin forms for measurement is often easier. However, in multivitamin supplements and milkbased formulations such as infant formula, vitamin premixes may be microencapsulated to promote long-term stability, and this formulation may complicate quantitative recovery. Vitamins may also be present in multiple forms that contribute to overall activity. For example, four naturally occurring tocopherol isomers contribute

S.A. Wise et al. / J. Chromatogr. A 1261 (2012) 3–22

13

Fig. 7. Comparison of results for the determination of PAHs, PCBs, and chlorinated pesticides using different internal standards in environmental-matrix SRMs.

to “vitamin E” in plant oils, but in processed foods and dietary supplements, ␣-tocopherol acetate may be used for fortification. Measurement methods must address the different chemical forms either through direct assays or by conversion of the species. Vitamins are subject to degradation by oxidation, exposure to light and heat, and isomerization, and analytical methods must consider these potential sources of bias.

Extraction Soxhlet extraction Pressurized Fluid Extraction (PFE) Use of Different Solvents

3.1. Development of independent methods for vitamins and carotenoids 3.1.1. Water-soluble vitamins LC is a well-established technique for the determination of water-soluble vitamins (WSVs), including the B vitamins and vitamin C, in food and dietary supplement matrices [71]. Because

Add 13C and/or deuterated PAHs as internal standards

Cleanup/Isolation SPE (silica, amino) LC (normal-phase) total PAH Fraction isomer Fraction

Separation and Detection LC-fluorescence (total fraction) LC-fluorescence (isomer fraction) GC-MS (different columns) 5% phenyl phase 50% phenyl phase Proprietary nonpolar phase 50% liquid crystalline phase

Fig. 8. Independent methods approach for value assignment of PAHs in environmental-matrix SRMs.

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A

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20

30

Time (min) Fig. 9. Comparison of two reversed-phase separations of water-soluble vitamins in SRM 3280 Multivitamin/Multielement Tablets. (A) C18 column (5 ␮m particle size) with an acetonitrile:phosphate buffer mobile phase with UV absorbance detection at 260 nm and (B) C18 column (3 ␮m particle size) with alternate selectivity with a methanol:ammonium formate buffer mobile phase with UV absorbance detection at 280 nm.

these compounds possess a variety of different structures, gradient elution is generally employed so that determination of multiple vitamins can be achieved in a single analysis. With the exception of pantothenic acid (vitamin B5 ), which lacks a suitable chromophore, the WSVs can be detected and quantified by monitoring UV absorbance at an appropriate wavelength [72]. LC–FL can also be used if the analyte has intrinsic fluorescence or can be converted to a form that fluoresces [73,74]. NIST has employed LC–UV and LC–FL methods for the separation and quantification of WSVs in a number of food and dietary supplement SRMs [75–77]. Separation of the vitamins is achieved by using reversed-phase LC on a C18 column with an aqueous-organic mobile phase. One challenge associated with the measurement of this group of analytes is that the WSVs are relatively polar and can be difficult to retain on traditional C18 phases. Also, in complex formulations, UV detection may not have sufficient specificity to ensure the absence of interferences. Because of the potential limitations of LC–UV approaches for determination of selected WSVs, and in an effort to develop a second independent analytical method for these compounds, NIST pursued development of a method based upon LC–MS for these analytes [78]. In LC–MS selected ion monitoring can provide enhanced specificity relative to UV absorbance detection, and multiple ions can be monitored in a single analysis. A second method was developed with an alternative separation and MS detection. The second method also utilizes a C18 column to achieve the desired separation, but the selectivity of this stationary phase for the analytes of interest is different, as shown in Fig. 9 [78]. For the LC–MS analysis, an ammonium formate buffer was used in place of a phosphate buffer. In addition, the LC–MS method takes advantage of changes in LC column technology, including the move toward

smaller particle diameters for column packing materials. Because the chromatograms in Fig. 9 were obtained using UV detection, no signal is visible for vitamin B5 in either chromatogram. However, through the use of selected ion monitoring in the LC–MS method, vitamin B5 was readily determined. An additional benefit using LC–MS methodology is that an isotope dilution approach can be employed for quantification through the use of stable isotope labeled internal standards for each of the vitamin analytes. Therefore, previously identified limitations of mass spectrometric methods, including potential matrix effects on ionization, were minimized, and excellent measurement precision was obtained. For the LC–MS method, labeled internal standards for vitamins B1 , B3 , B5 , and B6 were obtained and used for quantification. Both the LC–UV and LC–MS methods were applied to the value assignment of vitamins in two SRMs, SRM 3280 Multivitamin/Multielement Tablets and SRM 1849 Infant/Adult Nutritional Formula. SRM 3280 is similar in composition to commercial multivitamin tablets and contains both fat-soluble vitamins (FSVs) and WSVs as well as minerals and excipients that would commonly be found in multivitamin products [77]. Table 3 provides a comparison of the LC–UV and LC–MS results for WSVs in SRM 3280 [78]. The NIST LC–UV and LC–MS results were combined with data from collaborating laboratories to obtain the certified values shown in Table 3. A labeled internal standard was not available for vitamin B2 , and therefore this analyte was quantified using the labeled internal standard for vitamin B6 . As shown in Table 3, the agreement between the LC–UV and LC–MS methods is good, and measurement precision is comparable. The lack of a labeled internal standard may be responsible for the higher variability of the vitamin B2 results compared to the results for other analytes. Both LC–UV and LC–MS methods were also employed for value assignment of WSVs in SRM 1849, a milk-based powder fortified with vitamins and nutritional elements [76]. The concentration of the WSVs is approximately two orders of magnitude lower in SRM 1849 than in SRM 3280, and the high concentration of lipids in this SRM created some additional challenges for isolation of the WSVs prior to their measurement. Nevertheless, comparable results were obtained for the LC–UV and LC–MS methods, as shown in Table 3. The results obtained for SRM 3280 and SRM 1849 provide further evidence of the value of using complementary separation techniques and different detection schemes when assigning values to complex-matrix SRMs. 3.1.2. Fat-soluble vitamins Several independent methods have been developed for use in the certification of FSVs in food, dietary supplements, and human serum. Method independence is based on differences in the chromatographic separation and detection approaches. Methods typically target the measurement of FSVs and carotenoids in the same analysis, and to achieve this goal, LC column selection is a critical aspect of the work. Retinyl acetate or retinyl palmitate and ␣-tocopheryl acetate are relatively easy to separate from each other and from matrix constituents; however, baseline resolution of carotenoids is more challenging. Both monomeric and polymeric C18 columns have been used for the determination of FSVs, but better separation of carotenoid isomers is usually possible with the use of columns prepared with C30 polymeric surface modification chemistry [79–81] (see below). Separations of FSVs and carotenoids are illustrated in Fig. 10 for the analysis of SRM 3280 Multivitamin/Multielement Tablets using three different LC–UV methods [77,82]. The separation of an extract of SRM 3280 using a polymeric C18 column with isocratic elution and time-programmed absorbance detection is shown in Fig. 10A. The three wavelengths were optimized for the determination of retinyl acetate ( = 325 nm), ␣-tocopheryl acetate ( = 284 nm), and carotenoids ( = 450 nm). In Fig. 10B the

S.A. Wise et al. / J. Chromatogr. A 1261 (2012) 3–22

15

Table 3 Summary of results (mg/g) for water-soluble vitamins in SRM 3280 and SRM 1849. Relative standard deviations (% RSD) for the measurements are given in parentheses.

SRM 3280 Thiamine hydrochloride (B1 ) Riboflavin (B2 ) Niacinamide (B3 ) Pantothenic acid (B5 ) Pyridoxine hydrochloride (B6 ) SRM 1849 Thiamine chloride (B1 ) Riboflavin (B2 ) Niacinamide (B3 ) Pantothenic acid (B5 ) Pyridoxine hydrochloride (B6 ) a b

NIST LC–MS

NIST LC–UV

Certified valuea

1.17 (1.6) 1.47 (8.2) 13.9 (1.5) 8.1 (1.6) 1.86 (2.1)

1.06 (2.9) – 14.3 (1.7) – 1.75 (1.1)

1.06 1.32 14.10 7.30 1.81

± ± ± ± ±

0.12 0.17 0.23 0.96 0.17

15.2 (4.6) 16.6 (5.4) 98.8 (2.8) 66.0 (3.0) 14.0 (5.0)

15.2 (1.3) 18.1 (3.9) 98.4 (4.3) – 13.3 (3.0)b

15.8 17.4 97.5 64.8 14.2

± ± ± ± ±

1.3 1 2.3 2.2 1.5

Detailed description of the certification process and uncertainty for these two SRMs is provided in Refs. [76,77]. Vitamin B6 was determined using LC with fluorescence detection.

determination of trans-␤-carotene is shown using a C18 column with alternate selectivity and a ternary gradient for elution with absorbance detection at 450 nm. A polymeric C30 column, operated with gradient elution and detection at 450 nm was used for the determination of lutein and trans-␤-carotene (Fig. 10C). An LC/IDMS method was also developed for assessment of retinyl acetate, vitamin D2 , vitamin K1 , and ␣-tocopheryl acetate to complement

A

-Tocopherol (IS) -Tocopheryl acetate

Retinyl acetate

Trans- -carotene

Lutein

Cis- -carotene

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3

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40

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70

Time (min) Fig. 10. Methods used in the determination of fat-soluble vitamins and carotenoids in SRM 3280 Multivitamin/Multielement Tablets. (A) LC–UV with isocratic elution using a polymeric C18 column and time-programmed absorbance detection, 1 = 325 nm for retinyl acetate, 2 = 284 nm for tocopherols, and 3 = 450 nm for carotenoids; (B) LC–UV with ternary solvent mobile phase using an alternateselectivity C18 column and absorbance detection at 450 nm, and (C) LC–UV with gradient elution using a polymeric C30 column and absorbance detection at 450 nm. Adapted from reference [82]; see reference for chromatographic conditions.

the LC–UV methods (not shown). Stable isotope-labeled analogues were used as internal standards for retinyl acetate, vitamin D2 , and vitamin K1 .

3.1.3. Carotenoids Carotenoids are naturally occurring pigments that are found in a wide variety of plant and animal sources including most fruits and vegetables, certain animal tissues, and biological fluids. Although carotenoids are not (strictly speaking) classified as vitamins, the metabolism of ␤-carotene and certain other carotenoids results in the production of vitamin A. Carotenoid hydrocarbons are nonpolar, whereas hydroxy-substituted carotenoids, referred to as xanthophylls, are polar. The molecular structure of carotenoids is constrained by a network of conjugated double bonds, and carotenoid mixtures from natural sources contain both cis and trans forms. Methods for the determination of carotenoids have been developed using both normal-phase and reversed-phase LC. In a study of over 60 commercial C18 columns, better separations of carotenoids were usually achieved with polymeric C18 columns compared with monomeric C18 columns [83]. This trend is similar to that observed for other constrained-shape solutes, such as PAH isomers described above. To improve the separation of carotenoids, a new stationary phase was designed to exhibit enhanced shape recognition for carotenoid isomers. Based on a comparison of space-filling models of ␤-carotene with measurements of the thickness of a polymeric C18 stationary phase (≈2.8 nm vs. 2.1 nm), a new, thicker stationary phase was developed to permit more complete solute-stationary phase interactions. The resulting “carotenoid column” utilized a polymeric C30 surface modification synthesis which was optimized for separation of polar xanthophylls and nonpolar hydrocarbon carotenoids [79]. A silica substrate was selected with a pore size of 20 nm, which provided sufficient space for the extended length of the C30 phase and moderate specific surface area to provide adequate retention of the xanthophylls. The effect of endcapping was studied, and better separation of the xanthophylls resulted for columns that were not endcapped. A comparison of separations of carotenoid isomers is shown in Fig. 11 for a monomeric C18 column (A) and the polymeric C30 column (B). Both columns provide good separations of the polar carotenoids, but significantly improved resolution of the carotenes is achieved with the C30 column. As with other shape selective LC columns, further improvements in resolution are possible under subambient conditions [84]. Since NIST’s development of the carotenoid column in 1994, commercial sources of this product have become available, and there are numerous reports in the literature of their use for the determination of carotenoids [80].

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nonpolar carotenoids

A astaxanthin echininone

zeaxanthin lutein

-cryptoxanthin canthaxanthin

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80

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15-cis -carotene echininone

canthaxanthin zeaxanthin lutein

13-cis -carotene -carotene trans -carotene 9-cis -carotene

-cryptoxanthin

capsanthin astaxanthin

-carotene

0

10

20

30

40

50

lycopene

60

70

80

Time (min) Fig. 11. Separations of carotenoids and xanthophylls on (A) monomeric C18 column and (B) polymeric C30 column. Adapted from reference [79]; see reference for chromatographic conditions.

3.2. Development of independent methods for fatty acids The first food-matrix SRMs with values assigned for individual fatty acids were issued in the late 1990s based on measurements using GC–FID or GC–MS combined with results from collaborating laboratories. In the late 2000s the approach for assigning values for fatty acids at NIST changed significantly with the full implementation of two independent methods for measurements at NIST using different sample preparation methods followed by analysis using GC–FID and GC–MS. A summary of the SRMs developed to support measurements of fatty acids has been presented by Sander et al. [85]. The current implementation of the two independent methods approach for the determination of fatty acids in food and dietary supplements is illustrated in Fig. 12 using as examples two saw palmetto SRMs (SRM 3250 Serenoa repens Fruit and SRM 3251 Serenoa repens Extract) [86]. The extraction step used for the determination of fatty acids depends on the sample matrix. For SRM 3250 Serenoa repens Fruit, both Soxhlet extraction and PFE were used in a manner similar to that used for the environmental SRM certifications described above. However, for a different food matrix, SRM 1849 Infant/Adult Nutritional Formula, PFE was found not to quantitatively extract the fatty acids from matrix. Soxhlet extraction using a wetted diatomaceous earth substrate was necessary to free the fatty acids from the matrix [76]. Following extraction, the fatty acids in the extract are derivatized to produce fatty acid methyl esters (FAMEs). The two methods typically used for transesterification are a boron trifluoride method and a (m-trifluoromethylphenyl)trimethyl ammonium hydroxide method (see Fig. 12). For the food- and dietary-supplement matrix SRMs characterized for fatty acids, these two methods have been shown to be comparable. The final analysis steps for the fatty acid determinations have been GC–FID and GC–EI-MS; however, neither is particularly selective for the FAMEs, i.e., GC–FID is a universal detector for most carbon/hydrogen containing compounds, and the fatty acid methyl esters fragment in the source of the MS. To reduce this problem, a long (100 m), polar column (bis-cyanopropyl polysiloxane phase) has been used for the GC–FID analyses, and a shorter (60 m), relatively polar column (50% cyanopropyl/50% phenyl polysiloxane phase) has been used for the GC–MS analyses. These columns provide different separation selectivity for the fatty

acid methyl esters. The 50% cyanopropyl/50% phenyl polysiloxane phase retains the saturated fatty acid methyl esters longer relative to certain unsaturated fatty acids than the bis-cyanopropyl polysiloxane phase. For example, eicosanoic acid methyl ester elutes before ␣-linolenic acid methyl ester on the bis-cyanopropyl phase with the elution order reversed on the 50% cyanopropyl/50% phenyl phase, and docosanoic acid methyl ester elutes before arachidonic acid methyl ester and eicosapentaenoic acid (EPA) methyl ester on the bis-cyanopropyl phase with the elution order reversed on the 50% cyanopropyl/50% phenyl phase. The internal standards for these analyses have been deuterated fatty acids and/or fatty acids that are not present in the samples being analyzed, often odd-chain fatty acids. Since the number of deuterium atoms added to the fatty acids is large, the deuterated fatty acid methyl esters are baseline resolved from the native FAMEs on the columns used for these analyses. For the GC–MS analyses, it is particularly important to have each fatty acid being determined in the calibration solutions because the detector response varies for each fatty acid depending on its fragmentation in the MS. It is also important to monitor the detector response factors over time as fatty acids are prone to oxidation; thus the solutions used for calibration may degrade over time if not stored properly. 3.3. Development of independent methods for botanical dietary supplements Botanical dietary supplements are a recent category of naturalmatrix SRMs under development to support the needs of the dietary supplement industry. Recent federal regulations have imposed new measurement and reporting requirements on the manufacturers of dietary supplements, and reference materials have been developed to assist this community. In 1994, the U.S. Congress passed the Dietary Supplement Health and Education Act (DSHEA), which provided a separate legal status for dietary supplements and imposed certain requirements for the manufacture and sale of these materials [87]. More recently, the Food and Drug Administration issued current good manufacturing practices that in part require manufacturers to characterize the chemical composition of their ingredients and finished products [88]. Since 2002 NIST has been working

S.A. Wise et al. / J. Chromatogr. A 1261 (2012) 3–22

0.5 g Saw palmetto fruit

0.02 g Saw palmetto extract

add IS

add IS

PFE hexane/acetone (4:1 vol fraction)

0.5 g Saw palmetto fruit

17

0.25 g Saw palmetto extract

add IS

add IS

Soxhlet methylene chloride

BF3 methylation

Methylation using 0.1 mol/L methanolic (m-trifluoromethylphenyl)trimethyl ammonium hydroxide

GC-FID 100 m biscyanopropyl phase

GC/MS 60 m 50% cyanopropyl + 50% phenyl phase

Fig. 12. Analytical scheme for the determination of fatty acids in saw palmetto SRMs. Adapted from reference [86].

in collaboration with the National Institutes of Health, Office of Dietary Supplements and the U.S. Food and Drug Administration to develop SRMs to help support these measurement needs. Botanical dietary supplements offer unique measurement challenges related to the development of SRMs, and many of these challenges involve separation science. For the botanical dietary supplements, the compounds of interest, i.e., the active compound(s) or marker compound(s), are generally different for each material, e.g., adrenergic alkaloids in ephedra and bitter orange; flavonoids aglygones and terpene lactones (ginkgolides) in ginkgo; phytosterols and fatty acids in saw palmetto; catechins in green tea; anthocyanins and procyanidins in berries; and isoflavones in soy, kudzu, and red clover. The composition of manufactured products potentially includes native plant constituents (e.g., flavonoids, alkaloids, and species-specific biomarkers), environmental contaminants (e.g., pesticides, toxic elements), and additives (e.g., excipients, vitamins, minerals). Often dietary supplements are formulated using multiple plant species in highly complex mixtures composed of source materials from dried plant parts and/or dried extracts of plants. Because of the diverse nature of dietary supplements, corresponding reference materials are needed that are representative of the sample matrix. For this reason, SRM suites have been developed that include natural, extracted, and extensively processed forms of each botanical material. These SRM suites typically consist of dried, ground, and sieved plant parts; extract(s) of the plant that may be spray dried or otherwise converted to a dry powder form; and blend(s) of commercially available finished products. Measurement challenges include sample extraction and cleanup, and the development of selective separation and detection methods. Sample extraction methods for botanicals include liquid extraction with ultrasonic agitation (“sonication extraction”), liquid extraction with mechanical agitation, PFE, microwave extraction, and Soxhlet extraction. The cells in botanical matrices can inhibit release of plant constituents of interest. Quantitative recovery of biomarkers and other constituents of interest depend on this release; strategies include mechanical or chemical disruption of cell walls by grinding, freezing, enzymatic treatment, energy absorption, use of high pressure, or use of extended extraction times. The approaches selected must be balanced with the stability of the analytes (to light, heat, oxidation or other chemical

reactions). Because the recovery of a specific constituent from an unknown sample cannot be determined a priori, extraction studies are required to assess the validity of the method. To meet the requirement of method independence in the certification of marker compounds in botanical dietary supplements, multiple extraction steps in which individual extracts are combined are often required. Published, validated chromatographic separation methods for many of the active/marker compounds are limited, and if methods do exist, they are typically not based on mass spectrometry, thereby increasing the difficulty of implementing the requirement of two or more analytical methods for assigning certified values. Isotopically labeled analogues of the compounds of interest are almost nonexistent; thus hindering the development of ID GC–MS, ID LC–MS, or ID LC–MS/MS methods. To address the independent methods criteria for botanical dietary supplement SRMs, the focus has been on high-resolution separations using columns of differing selectivity combined with selective detection. The implementation of the independent chromatographic methods approach for dietary supplements is described below for two botanical dietary supplement materials, ephedra and ginkgo biloba. Additional examples of multiple methods for botanical dietary supplements include bitter orange [89], saw palmetto [86,90], green tea [91], and berries [92]. 3.3.1. Ephedra Ephedra was identified as the first priority for development of botanical dietary supplement SRMs based on safety concerns for users, and a suite of ephedra-containing SRMs was issued to support the accuracy of label claims for these products [93,94]. In 2003 the FDA issued a ruling that effectively banned sales of ephedracontaining dietary supplements in the U.S. Prior to this ruling, such dietary supplements were widely marketed as aids for weight loss and as stimulants to promote energy. Although the need for ephedra-containing SRMs no longer exists and the materials are no longer available, the methods of analysis that were developed for their certification provided valuable experience and a model for the use of independent measurement approaches in the certification of the other botanical dietary supplement SRMs that followed. The powdered botanical Ephedrae herba is a traditional Chinese medicine that is used to treat a variety of ailments. Over 50 species of ephedra are known, and about 18 species contain significant levels of ephedrine alkaloids. The activity of this plant is attributed

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to six ephedrine diastereomers (12 chiral isomers); the most abundant naturally occurring form is (−)-ephedrine. Five ephedracontaining SRMs were developed: SRM 3240 Ephedra sinica Stapf Aerial Parts, SRM 3241 Ephedra sinica Stapf Native Extract, SRM 3242 Ephedra sinica Stapf Commercial Extract, SRM 3243 Ephedra – Containing Solid Oral Dosage Form, and SRM 3244 Ephedra – Containing Protein Powder. Four methods were developed at NIST to assign values for the ephedrine alkaloids in these materials: LC–UV (method 1), LC–MS (method 2), LC–MS/MS (method 3), and CE (method 4). Representative separations for these methods are provided in Fig. 13 for the analysis of SRM 3243. Method 1 (Fig. 13A) was based on the work of Roman [95] with minor modifications to the chromatographic separation and quantification using terbutaline as an internal standard. Because ephedrine alkaloids contain weak UV chromophores, relatively nonselective absorbance detection was required at 208 nm, and matrix interferences were apparent. In most cases, these interferences were not significant since levels of the alkaloids ranged from about 0.1 mg/g to 30 mg/g. Method 2 (Fig. 13B) utilized a different phenyl column and mobile phase conditions compared with those in method 1, and quantification was based on the addition of ephedrine-d3 as an internal standard. Matrix interferences were not apparent in the selected ion chromatograms (positive ion, ESI-MS). Method 3 used a chromatographic separation similar to that used in method 2. Transitions for multiple reaction monitoring (MRM) are indicated in Fig. 13C. Better sensitivity was obtained by this method, and more reliable quantification was possible for the low level of methylpseudoephedrine. Chiral separations of the major alkaloids ephedrine and pseudoephedrine were achieved by using capillary electrophoresis using cyclodextrin-based chiral selectors (method 4, Fig. 13D). This method was developed to verify that synthetic adulterants were not present in any of the SRM materials (naturally occurring ephedrine alkaloids are present in botanicals in non-racemic, chiral forms). In addition to measurements made at NIST, alkaloids were determined by three collaborating laboratories. Some of the methods used by the collaborating laboratories were similar to methods used at NIST; however, a unique method was developed by National Research Council Canada based on high-field asymmetric waveform ion mobility spectrometry (FAIMS). This method did not employ a chromatographic separation; instead the measurements were made using flow injection and a highly specific mass spectrometry technology, thereby providing a method independent of the chromatographic separation step. The details of the value assignment process for the ephedra materials, including the comparison of the results from the different methods, are described in Sander et al. [93]. 3.3.2. Ginkgo biloba Ginkgo leaves and extracts are used in the formulation of dietary supplements with perceived health benefits for memory improvement and cognition enhancement. A suite of three SRMs was developed to provide sample matrices representative of industry needs: SRM 3246 Ginkgo biloba (Leaves), SRM 3247 Ginkgo biloba Extract, and SRM 3248 Ginkgo-Containing Tablets [96]. Two types of constituents are commonly measured in ginkgo-containing dietary supplements, flavonol glycosides, and terpene lactones. The flavonol glycosides are hydrolyzed, and the flavonols are determined by LC–UV or LC–MS (see Fig. 14A) [96]. Separation of the flavonols was readily achieved using a reversed-phase gradient with a C18 column. Two wavelengths were monitored, 287 nm and 370 nm. Hesperitin was selected as an internal standard and was monitored at 287 nm. The flavonols (quercetin, isorhamnetin, and kaempferol) were monitored at 370 nm. This dual-wavelength approach reduced matrix interferences for the three flavonols, and permitted detection of the internal standard. Slightly modified

NPE

A

E PE

NE

IS

0

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10

20

Time (min) m/z = 150

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Synephrine

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Time (min) Fig. 13. Complementary methods used in the certification of SRM 3243 EphedraContaining Solid Oral Dosage Form. (A) LC–UV with isocratic elution using a alkyl phenyl column and detection at 208 nm, (B) LC–MS with isocratic elution using a phenyl column and positive ion ESI-MS, (C) LC–MS/MS with similar separation conditions as in 1B and with precursor and fragment ions as indicated, and (D) capillary electrophoresis using three different cyclodextrin-based chiral selectors and absorbance detection at 210 nm. Component identification: m/z 134 norephedrine (NE) and norpseudoephedrine (NPE); m/z 148 ephedrine (E) and pseudoephedrine (PE); m/z 180 methylephedrine (ME) and methylpseudoephedrine (MPE). Adapted from reference [93]; see reference for chromatographic conditions.

S.A. Wise et al. / J. Chromatogr. A 1261 (2012) 3–22

K

A

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Relative response

I

= 287 nm

C12 column, LC/ESI-MS L (IS)

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Time (min) Fig. 14. Comparison of detection approaches for the measurement of flavonoids in SRM 3248 Ginkgo-Containing Tablets. (A) LC–UV with gradient elution using a C18 column with detection at 287 nm (upper chromatogram) and 370 nm (lower chromatogram), and (B) LC–MS with similar separation conditions as in 4A and with positive ion ESI-MS. Selected ions: m/z 303 quercetin (Q) and hesperetin (H) (IS = internal standard); m/z 317 isorhamnetin (I); and m/z 287 kaempferol (K). Adapted from reference [96]; see reference for chromatographic conditions.

separation conditions were used with the LC–MS method, and the four compounds were easily detected by positive ion ESI-MS with no apparent matrix interferences. Methods were also developed for the determination of terpene lactones (ginkgolides and bilobalide) in the ginkgo-containing SRMs based on independent chromatographic separations and different MS ionization approaches (see Fig. 15). In the first method, sample extracts were analyzed using a C12 column with a methanol/water gradient and positive ion ESIMS with selected ion monitoring. Different separation selectivity was achieved in a second method using a C18 column with acetonitrile/water gradient with APCI-MS detection. Different ions were monitored in the two MS methods, and different internal standards were employed for added independence. 3.4. Development of independent methods for clinical health status markers NIST’s involvement in the development of reference methods and reference materials to support accuracy in clinical diagnostics spans more than four decades [97]. This effort began with the introduction of high-purity standards for clinical analytes such as glucose and cholesterol and gradually expanded to include serum matrix-based reference materials that are intended to mimic the types of samples encountered in clinical laboratories. Although NIST generally employs multiple independent methods for value assignment of natural-matrix reference materials, clinical SRMs represent an area where a single higher-order method has been used for certification measurements. These higher-order methods were originally known as definitive methods, although the terms reference measurement procedure or reference methods are now

Fig. 15. Methods used in the measurement of ginkgolides in SRM 3248 GinkgoContaining Tablets. (A) LC–MS with gradient elution using a C12 column and positive ion ESI-MS; (B) LC–MS with gradient elution using a C18 column and negative ion APCI-MS. Peak identities are: bilobalide (BB), ginkgolide J (G–J), ginkgolide C (G–C), ginkgolide A (G–A), ginkgolide B (G–B), limonin (L), and hesperetin (H); internal standard (IS). Selected ions for ESI-MS: m/z 344 (BB), m/z 426 (G-A), m/z 442 (G–J) and (G–B), m/z 458 (G–C), m/z 488 (L). Selected ions for APCI-MS: m/z 325 (BB), m/z 467 (G–A), m/z 423 (G–J) and (G–B), m/z 439 (G–C), m/z 301 (H), m/z 483 (G–J). Adapted from reference [96]; see reference for chromatographic conditions.

used to describe methods of this caliber. In order to serve as standalone approaches for value assignment, these methods need to be rigorously evaluated, and criteria for acceptance have been developed [98,99]. During the 1980s and 1990s, NIST developed a suite of definitive methods for clinical analytes such as creatinine, glucose, and uric acid in serum [100–102]. These methods were based upon GC–MS and utilized isotope dilution approaches for analyte quantification. In these methods, derivatization of the analyte was frequently necessary to convert it into a form amenable to GC analysis. For example, the determination of glucose by GC–MS involved the use of a butylboronic acid derivative [101,103], and a tert-butyldimethylsilyl derivative was used for uric acid [102]. Derivatization also yields higher mass ions which reduces the risk of interferences. Although capillary GC–MS provides a high peak capacity, there is still a possibility that an interfering species could coelute with the analyte of interest and contribute to the measured ion intensity. Therefore, confirmatory measurements were often performed using a GC column having different polarity [104,105]. In some cases, different ions were also monitored for these confirmatory measurements. Although these measurements were not generally used in the SRM value assignment, the agreement between this confirmatory data and the primary measurement results provided additional confidence that no significant measurement bias existed. In recent years, the use of GC–MS for value assignment of clinical SRMs has diminished. These methods tend to be labor intensive because of the steps needed to isolate the analyte from the serum matrix and the need for derivatization. Both LC–MS and LC–MS/MS have begun to replace GC–MS for SRM certification measurements. For some clinical analytes, a simple protein precipitation is sufficient to release the analyte from the matrix prior to LC–MS or LC–MS/MS analysis. In addition, derivatization is generally not required, although it may be employed to improve sensitivity [106]. The development of SRM 967 Creatinine in Frozen Human

20

S.A. Wise et al. / J. Chromatogr. A 1261 (2012) 3–22

Table 4 Comparison of creatinine concentrations determined in SRM 967 by using GC–MS and LC–MS. GC–MS method

Mean SD CV (%)

LC–MS method

Pool 1 (␮mol/L)

Pool 2 (␮mol/L)

Pool 1 (␮mol/L)

Pool 2 (␮mol/L)

67.0 0.6 0.9

346.1 1.6 0.45

66.1 0.2 0.2

346.3 0.8 0.2

30 LC-MS/MS GC-TOF-MS (MTBSTFA) GCxGC-TOF-MS (MTBSTFA) GC-TOF-MS (PCF)

25

Mass Fraction ( g/g)

20

15

10

5

L-valine

L-tyrosine

L-threonine

L-serine

L-proline

L-phenylalanine

L-ornithine

L-methionine

L-lysine

L-leucine

L-isoleucine

L-histidine

glycine

L-glutamic acid

L-cystine

L-cysteine

L-arginine

L-alanine

0

Fig. 16. Comparison of results from different methods for the determination of amino acids in SRM 1950 Metabolites in Human Plasma. MTBSTFA, N-methyl-N-(tertbutyldimethylsilyl)trifluoroacetamide; PCF, propyl chloroformate. Adapted from reference [108].

Serum provides an example of the transition that is underway from ID GC–MS to ID LC–MS(/MS) for value assignment measurements [107]. Creatinine is present in serum at concentrations near 1.0 mg/dL in healthy individuals and increases in individuals suffering from chronic kidney disease. Therefore, SRM 967 is comprised of two pools of human serum, one with a normal concentration of creatinine and the other with an elevated concentration. The original NIST GC–MS definitive method for creatinine was developed in the 1980s and includes the use of an ion-exchange resin to isolate creatine from creatinine because these compounds will form the same derivative [100]. Freeze-drying of the sample extracts is also necessary to exclude water prior to the derivatization step. In the LC–MS method, sample preparation is basically limited to a protein precipitation step, and any creatine present is resolved chromatographically on a C18 column during the LC–MS analysis [107]. Ions for both creatinine and creatine can be monitored during the analysis. Table 4 shows a comparison of the results obtained by GC–MS and LC–MS for the two serum pools that comprise SRM 967. As shown in Table 4, the results are in excellent agreement, and based upon the favorable outcome of this comparison, NIST now employs the LC–MS method routinely for the determination of creatinine in serum and plasma. Although LC–MS and LC–MS/MS methods have begun to dominate the value assignment process for clinical SRMs, there are cases where GC–MS and LC–MS/MS can complement one another. Amino acids represent an important class of metabolites, and their

concentrations in serum, plasma, or urine can be used in the diagnosis of a variety of different diseases. Therefore, we elected to include the determination of amino acids in the development of SRM 1950 Metabolites in Human Plasma. Four different strategies were utilized for measurement of the amino acids in plasma, with three based upon GC–TOF-MS or GC × GC–TOF-MS and the fourth based upon LC–MS/MS [108]. Two different derivatization strategies were employed for the GC–MS analyses. In most cases, the results were comparable across the techniques, as shown in Fig. 16, although some differences were noted. For example, the derivative of histidine was not sufficiently resolved from other constituents by GC–TOF-MS to permit accurate determination, but incorporation of a second separation dimension (GC × GC) resulted in complete chromatographic resolution and improved measurement confidence. In some cases, an amino acid could only be determined reliably by LC–MS/MS, and not by any of the GC–MS platforms, as was the case for arginine and ornithine. Under the temperature conditions employed in the GC–MS approaches, conversion of arginine to ornithine could occur; therefore LC–MS/MS is a better choice for these analytes [108]. 4. Conclusions Separation science plays a critical role in the development and implementation of the multiple independent methods approach used at NIST for the certification of SRMs, particularly the natural

S.A. Wise et al. / J. Chromatogr. A 1261 (2012) 3–22

matrix SRMs with values assigned for trace organic constituents. It was perhaps fortunate that when the first SRMs for organic constituents were developed over 30 years ago, the focus was on the determination of PAHs, a class of contaminants that contained numerous isomers and that could be determined using both LC and GC. The requirement to implement two or more methods was a primary motivation to investigate and to better understand the differences in chromatographic selectivity among different stationary phases in both GC and LC. With the advent of the highly sensitive and selective detection capabilities provided by LC/MS, and particularly LC/MS/MS, the emphasis on improvements in the chromatographic separation (both through high resolution and selectivity) has perhaps been diminished within the organic analytical measurement community. However, as long as NIST pursues the use of multiple independent methods as a means of minimizing measurement bias, the understanding of separation science will remain at the forefront in the development of SRMs for organic analysis. Acknowledgments The authors acknowledge the contributions of numerous analysts within the Analytical Chemistry Division and outside collaborators who over the past 30 years have contributed to the development of methods and provided measurements used for the certification of the natural-matrix SRMs described in this review. The following NIST analysts contributed to methods and results described in this review: Mary Bedner, Holly A. Bamford, Nathan G. Dodder, Samuel Howerton, Jennifer M. Keller, John R. Kucklick, Mark S. Lowenthal, Elizabeth A. McGaw, Melissa M. Phillips, Dianne L. Poster, Catherine A. Rimmer, Mary B. Satterfield, Katherine E. Sharpless, Jeanice B. Thomas, and Michael J. Welch. The following guest scientists working at NIST also contributed to these SRMs: Angela Deissler, Toshihide Ihara, Maria J. Lopez de Alda, and Patricia Schubert. References [1] Bureau International des Poids et Mesures, Pavillon de Breteuil F-92312 Sevres Cedex France, 2012. [2] W.F. Hillebrand, J. Ind. Eng. Chem. 8 (1916) 466. [3] M. Epstein, Spectrochim. Acta 46B (1991) 1583. [4] H.S. Hertz, J.M. Brown, S.N. Chesler, F.R. Guenther, L.R. Hilpert, W.E. May, R.M. Parris, S.A. Wise, Anal. Chem. 52 (1980) 1650. [5] W. May, R. Parris, C. Beck, J. Fassett, R. Greenberg, F. Guenther, G. Kramer, S. Wise, T. Gills, J. Colbert, R. Gettings, B. MacDonald, NIST Spec. Publ. 260-136, National Institute of Standards and Technology, U.S. Government Printing Office, Gaithersburg, MD, 2000. [6] W.F. Kline, S.A. Wise, W.E. May, J. Liq. Chromatogr. 8 (1985) 223. [7] W.E. May, S.A. Wise, Anal. Chem. 56 (1984) 225. [8] S.A. Wise, B.A. Benner, S.N. Chesler, L.R. Hilpert, C.R. Vogt, W.E. May, Anal. Chem. 58 (1986) 3067. [9] S.A. Wise, B.A. Benner Jr., G.D. Byrd, S.N. Chesler, R.E. Rebbert, M.M. Schantz, Anal. Chem. 60 (1988) 887. [10] M.M. Schantz, B.A. Benner, S.N. Chesler, B.J. Koster, K.E. Hehn, S.F. Stone, W.R. Kelly, R. Zeisler, S.A. Wise, Fresenius’ J. Anal. Chem. 338 (1990) 501. [11] S.A. Wise, M.M. Schantz, B.A. Benner Jr., M.J. Hays, S.B. Schiller, Anal. Chem. 67 (1995) 1171. [12] S.A. Wise, B.A. Benner Jr., R.G. Christensen, B.J. Koster, J. Kurz, M.M. Schantz, R. Zeisler, Environ. Sci. Technol. 25 (1991) 1695. [13] S.A. Wise, W.J. Bonnett, F.R. Guenther, W.E. May, J. Chromatogr. Sci. 19 (1981) 457. [14] S.A. Wise, L.C. Sander, J. High Resolut. Chromatogr. Chromatogr. Commun. 8 (1985) 248. [15] S. Bachmann, C. Hellriegel, J. Wegmann, H. Handel, K. Albert, Solid State Nucl. Magn. Res. 17 (2000) 39. [16] S.A. Wise, S.N. Chesler, H.S. Hertz, L.R. Hilpert, W.E. May, Anal. Chem. 49 (1977) 2306. [17] S.A. Wise, L.R. Hilpert, R.E. Rebbert, L.C. Sander, M.M. Schantz, S.N. Chesler, W.E. May, Z. Fresenius’, Anal. Chem. 332 (1988) 573. [18] M.L. Lee, B.W. Wright, J. Chromatogr. 184 (1980) 235. [19] P.A. Peaden, B.W. Wright, M.L. Lee, Chromatographia 15 (1982) 335. [20] R.C. Kong, M.L. Lee, Y. Tominaga, R. Pratap, M. Iwao, R.N. Castle, Anal. Chem. 54 (1982) 1802.

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