Method 8276 8276 - 1 Revision 1 

 July 2014 

METHOD 8276

TOXAPHENE AND TOXAPHENE CONGENERS BY GAS CHROMATOGRAPHY/NEGATIVE ION 

CHEMICAL IONIZATION MASS SPECTROMETRY (GC-NICI/MS) 

SW-846 is not intended to be an analytical training manual. Therefore, method procedures 

are written based on the assumption that they will be performed by analysts formally trained in the 

basic principles of chemical analysis and in the use of the subject technology. 

In addition, SW-846 methods, with the exception of required use for the analysis of 

method-defined parameters, are intended to be guidance methods which contain general 

information on how to perform an analytical procedure or technique, which a laboratory can use 

as a basic starting point for generating its own detailed standard operating procedure (SOP), 

either for its own general use or for a specific project application. Performance data included in 

this method are for guidance purposes only, and must not be used as absolute quality control 

(QC) acceptance criteria for the purposes of laboratory QC or accreditation. 

1.0 SCOPE AND APPLICATION 

 Please see Appendix A at the back of this document for a summary of revisions to Method 

8276 (From March 2010). 

1.1 This method is used to determine the concentrations of various toxaphene 

congeners (see table below) and technical toxaphene (along with the possible addition of other 

toxaphene congeners and compounds from Method 8081) in extracts from solid and liquid 

matrices, using fused-silica, open-tubular capillary columns with negative ion chemical ionization 

mass spectrometry (NICI/MS). The approach emphasizes the analytical conditions recommended 

for technical toxaphene and for toxaphene congeners as compared to weathered toxaphene. 

Technical toxaphene can be definitively quantitated by NICI/MS while weathered toxaphene may 

only appear to be present based on the detection of ions found in toxaphene or the presence of 

known degradation products of toxaphene (e.g., Hx-Sed and Hp-Sed). For this reason, the 

quantitation of weathered toxaphene may be considered subjective and qualitative with the 

success highly dependent on matching the calibration standards to the weathered peak pattern. 

For clarification, any notation of toxaphene in the method hereafter will refer to technical 

toxaphene. 

Compound CAS Registry No.a

Toxaphene 8001-35-2 

Toxaphene Congeners: 

2-exo,3-endo,6-exo,8,9,10-Hexachlorobornane (Hx-Sed) 57981-29-0 

2-endo,3-exo,5-endo,6-exo,8,9,10-Heptachlorobornane (Hp-Sed) 70649-42-2 

2-endo,3-exo,5-endo,6-exo,8,8,10,10-Octachlorobornane (P26) 142534-71-2 

2-endo,3-exo,5-endo,6-exo,8,9,10,10-Octachlorobornane (P40) 166021-27-8 

2-exo,3-endo,5-exo,8,9,9,10,10-Octachlorobornane (P41) 165820-16-6 

2-exo,5,5,8,9,9,10,10-Octachlorobornane (P44) 165820-17-7 

2-endo,3-exo,5-endo,6-exo,8,8,9,10,10-Nonachlorobornane (P50) 6680-80-8 

2,2,5,5,8,9,9,10,10-Nonachlorobornane (P62) 154159-06-5 

a Chemical Abstract Service Registry Number

 
Method 8276 8276 - 2 Revision 1 

 July 2014 

1.2 The analyst should select gas chromatography (GC) columns, detectors, and 

calibration procedures most appropriate for the specific analytes of interest in a particular 

project application. Matrix-specific performance data should be generated, and the stability of 

the analytical system and instrument calibration must be established for each analytical matrix 

(e.g., hexane solutions from various sample matrix extractions). Example chromatograms and 

GC-NICI/MS conditions are provided as guidance. 

1.3 Although performance data are presented only for toxaphene and toxaphene 

congeners, in future method revisions additional target analytes (e.g., from Method 8081) may 

be added if acceptable performance can be demonstrated. When more analytes are included, it 

may become likely that not all of them can be determined in a single analysis. The chemical and 

chromatographic behaviors of these additional chemicals may result in coelution of some target 

analytes. Several cleanup/fractionation schemes are provided in this method for additional 

details refer to Method 3600. 

1.4 Multi-component mixtures (e.g., chlordane and toxaphene) present additional 

difficulties that include the need to separate congeners (Sec. 2.5 and 3.1) of the mixture. When 

samples contain more than one multi-component analyte, a higher level of expertise is 

necessary to attain acceptable levels of qualitative and quantitative analysis. The same is true 

of multi-component analytes that have been subjected to degradation by the environment or 

treatment technologies. These processes result in “weathered” multi-component mixtures that 

may have significant differences in peak patterns compared to those of the standards. 

1.5 Unless present in relatively high concentrations, 4,4’-DDD, 4,4’-DDE, 4,4’-DDT 

and related compounds will not pose significant interferences to toxaphene analysis by NICI/MS 

because they do not respond with similar sensitivity. 

1.6 This method has primarily been validated for the analysis of target analytes listed 

in Sec. 1.1. Extracts suitable for analysis by this method may also be analyzed for other 

organochlorine and organophosphorus pesticides (Methods 8081 and 8141) provided 

acceptable performance data can be generated. Additionally, some extracts may also be 

suitable for triazine herbicide analysis. However, low recoveries of triazine herbicides may result 

from lack of sample preservation. In addition, should users generate acceptable performance 

data for organochlorine pesticide compounds as noted in Method 8081, this method may be 

considered as an appropriate alternative to Method 8081. 

1.7 Prior to employing this method, analysts are advised to consult the base method 

for each type of procedure that may be employed in the overall analysis (e.g., Methods 3500, 

3600, and 8000) for additional information on QC procedures, development of QC acceptance 

criteria, calculations, and general guidance. Analysts also should consult the disclaimer 

statement at the front of the SW-846 manual and the information in Chapter Two for 1) guidance 

on the intended flexibility in the choice of methods, apparatus, materials, reagents, and 

supplies; and 2) the responsibilities of the analyst for demonstrating that the techniques 

employed are appropriate for the target analytes in the matrix of interest, and at the level(s) of 

concern. 

In addition, analysts and data users are advised that, except where explicitly specified in 

a regulation, the use of SW-846 methods is not mandatory in response to Federal testing 

requirements. The information contained in this method is provided by the Environmental 

Protection Agency (EPA) as guidance to be used by the analyst and the regulatory community 

in making judgments necessary to generate results that meet the data quality objectives (DQOs) 
Method 8276 8276 - 3 Revision 1 

 July 2014 

for the intended application. 

1.8 Application of this method is restricted to use by, or under the supervision of, 

personnel appropriately experienced and trained in the use of GC-NICI/MS and skilled in the 

interpretation of applicable chromatograms. Each analyst must demonstrate the ability to 

generate acceptable results with this method. 

1.9 This method depends on resonance electron capture (electron attachment) or 

dissociative electron capture in the ion source when the instrument is operated under methane 

chemical ionization (CI) conditions. Thus, the technique produces a transient ionic species that 

can be detected as a molecular anion of a compound or as a dissociated ion of that species 

produced by dissociative electron capture. Other moderating gases besides methane may be 

used provided acceptable project-specific performance data can be generated. Within this 

description, the technique does not strictly result from a CI event (i.e., the result of an ionmolecule reaction) and refers to the production of negative ions by any of these processes as 

NICI/MS. See Ref. 1 for a more detailed discussion of NICI/MS theory. 

2.0 SUMMARY OF METHOD 

2.1 The samples are prepared for analysis by GC-NICI/MS using the 

appropriate sample preparation (refer to Method 3500) and, if necessary, sample 

cleanup procedures (refer to Method 3600). 

2.2 The extract is analyzed by injection of a sample aliquot into a gas chromatograph 

with a narrow-bore fused-silica capillary column interfaced to a mass spectrometer capable of 

performing NIMS under CI conditions with methane as the introduced gas. 

2.3 Analysis of toxaphene involves monitoring a series of ions representing various 

congener groups found in the mixture and integrating all of these signals for a total toxaphene 

response. In the case of the toxaphene congeners, individual compounds are quantitated 

separately and reported separately. 

2.4 This method does not address all the congeners that comprise toxaphene. 

Toxaphene congeners can be added to the method by identifying retention windows with the 

additional congeners, assuming their responses are resolved from other congeners. Any added 

analytes must meet performance-based QC acceptance criteria. 

3.0 DEFINITIONS 

3.1 Toxaphene - A complex mixture of polychlorinated monoterpenes (primarily 

bornanes and camphenes) produced commercially from 1947 to 1982 and purported to 

contain 600-plus separate congeners. Also known as camphechlor, Melipax, chlorocamphene, 

polychlorocamphene, and chlorinated camphene. 

3.2 GC-NICI/MS - All techniques producing negatively charged ions used for 

confirmation and quantitation of analytes. See Sec. 1.9 for additional details. 

3.3 Congener - One of many variants or configurations of a common starting 

material. Most often the term is used to describe a chlorinated or brominated species of a 
Method 8276 8276 - 4 Revision 1 

 July 2014 

given starting material that gives rise to multiple levels of halogenation to fully halogenated 

compounds. 

3.4 Parlar number - Individual toxaphene congeners based on GC elution order are 

also known as "Parlars". For ease of reference, specific congeners have been given a Parlar 

number (e.g., P26, P50). More information on Parlar nomenclature can be found in reference 

28. 

3.5 Weathered toxaphene - In the environment, technical toxaphene degrades over 

time via both biotic and abiotic processes. The major processes of this degradation seem to be 

dechlorination and dehydrochlorination that lead to a pronounced shift toward lower chlorinated 

homologs. Weathered toxaphene may have a different chromatographic profile and NICI/MS 

detector response when compared to “virgin” toxaphene; this is a result of the composition 

changes which occurs from environmental degradation. 

3.6 Total Ion Current (TIC) - The sum of the separate ion currents carried by 

the different ions contributing to the spectrum (sometimes called the reconstructed ion 

current). 

3.7 Refer to Chapter One and the instrument manufacturer's instructions for 

additional definitions that may be relevant to this procedure. 

4.0 INTERFERENCES 

4.1 Solvents, reagents, glassware, and other sample processing hardware may yield 

artifacts and/or interferences during sample analysis. All of these materials must be 

demonstrated to be free from interferences under conditions of analysis by method blanks. 

Specific selection of reagents may be necessary. Refer to each method used for specific 

guidance on QC procedures and to Chapter Four for general guidance on glassware cleaning. 

Also refer to Methods 3500, 3600, and 8000 for a discussion of interferences. 

4.2 Interferences co-extracted from the samples will vary considerably from matrix to 

matrix. While general cleanup techniques are referenced or provided as part of this method, 

unique samples may require additional cleanup approaches to achieve desired degrees of 

discrimination and quantitation. Sources of interference in this method can be grouped into three 

broad categories as follows. 

4.2.1 Contaminated solvents, reagents, or sample processing hardware 

4.2.2 Contaminated GC carrier gas, parts, column surfaces, or detector 

surfaces 

4.2.3 Compounds extracted from the sample matrix to which the detector will 

respond 

4.3 Interferences by phthalate esters introduced during sample preparation can pose 

a major problem in some pesticide determinations but not for toxaphene analysis provided the 

scan window or ion monitoring signal is greater than m/z 300. However, extremely high levels of 

phthalate contamination could adversely affect quantitative responses of coeluting target 

components by reducing the availability of thermal electrons or reagent ions needed to ionize 
Method 8276 8276 - 5 Revision 1 

 July 2014 

target analytes. Interferences from phthalate esters can best be minimized by avoiding contact 

with any plastic materials and checking all solvents and reagents for phthalate contamination. 

4.3.1 Common flexible plastics contain varying amounts of phthalate esters 

that are easily extracted or leached from such materials during laboratory operations. 

4.3.2 Exhaustive cleanup of solvents, reagents, and glassware may be 

necessary to eliminate background phthalate ester contamination. 

4.3.3 Phthalate esters may be removed prior to analysis using Method 3640 

(GPC) or Method 3630 (silica gel cleanup). 

4.3.3 Cross-contamination of clean glassware routinely occurs when plastics 

are handled during extraction steps, especially when solvent-wetted surfaces are 

manipulated. 

4.4 The presence of sulfur will result in broad peaks that interfere with the detection 

of early-eluting compounds only if the monitored ions occur near m/z 256, 224, 192, 160, 128, 

and 96 (as well as the M+2 isotope due to 34S and the corresponding M+1 peaks from 33S). 

Therefore, sulfur should not interfere with monitored ions from toxaphene but should 

nevertheless be removed because of deleterious effects on chromatography and on the 

response of the NICI/MS ion source during coelution. Sulfur contamination should be expected 

with sediment samples. 

Method 3660 is suggested for removal of sulfur. Because the recovery of endrin 

aldehyde is drastically reduced when using the tetrabutylammonium (TBA) procedure in Method 

3660, it must be determined prior to sulfur cleanup if it is an analyte of interest. Endrin aldehyde 

is not affected by copper powder, so it can be determined after the removal of sulfur by using 

the copper powder technique in Method 3660. However, as indicated in Method 3660, copper 

powder may adversely affect the recoveries of other potential analytes of interest, including 

some organochlorine compounds and many organophosphorus compounds. 

4.5 Waxes, lipids, and other high molecular weight materials can be removed by 

Method 3640 (GPC cleanup) but are generally transparent to NICI/MS. Naturally occurring 

compounds such as flavonoids (e.g., coumarins) that contain conjugated carbonyl groups may 

respond sensitively under GC-NICI/MS conditions as will other kinds of compounds containing 

functional groups that facilitate ionization under these conditions. 

4.6 Other halogenated pesticides or industrial chemicals may interfere with the 

analysis of pesticides. Certain coeluting organophosphorus pesticides may be eliminated using 

Method 3640 (pesticide option). Coeluting chlorophenols may be removed with Methods 3630 

(silica gel), Method 3620 (Florisil®), or Method 3610 (alumina). 

4.7 PCB Interference 

4.7.1 Polychlorinated biphenyls (PCBs) also may interfere with the analysis of 

organochlorine pesticides. The problem may be most severe for the analysis of 

multicomponent analytes such as chlordane, toxaphene, and Strobane. If PCBs are 

known or expected to occur in samples, the analyst should consult Methods 3620 and 

3630 for techniques that may be used to separate the pesticides from the PCBs. Under 

NICI/MS conditions in the absence of oxygen (oxygen reacts with PCBs), PCBs should 
Method 8276 8276 - 6 Revision 1 

 July 2014 

not constitute an interference concern for toxaphene ions. The presence of the oxygen 

reaction does not preclude analysis, but must lead to reproducible results and would 

require removal of PCBs from extracts prior to toxaphene analysis. Overall, the success 

of analysis is strongly dependent on adequate cleanup, separations of 

congeners/analytes, and quantitative recoveries. 

4.7.2 Based on limited studies to date, the oxygen reaction observed with 

PCBs that gives rise to ions potentially interfering with toxaphene determination (i.e., 

ions at the same nominal mass but not the same elemental composition) is completely 

eliminated in modern instruments under appropriate conditions. The use of a PCB 

congener (e.g., PCB #204) serves to monitor this situation by the absence of (M - Cl + 

O)─ ions (e.g., m/z 411) at its retention time (less than 0.5% possible attributable 

response). Thus the success of this method depends heavily on the proper extraction, 

cleanup, concentration, separations, and mass spectrometric conditions to achieve the 

quantitation and confirm the target analytes. 

4.8 Coelution among the target analytes in this method can cause interference 

problems. Toxaphene congener coelution may be a problem for all possible GC columns 

regardless of the proposed analysis scheme. 

4.9 Chlordane interference 

4.9.1 Under GC-NICI/MS conditions, the determination of toxaphene and 

individual congeners of toxaphene may be affected by the presence of chlordane. In 

addition, chlordane congeners may coelute with certain toxaphene congeners and the13C 

isotope of the chlordane ion may add intensity to the corresponding isobaric ion of 

toxaphene. 

4.9.2 As shown in Table 3, ions for monitoring the presence of chlordane 

indicate that their 13C isotopes can contribute signals to monitored toxaphene ions. 

Therefore, use Table 3 to confirm whether chlordane is present at levels which 

significantly affect toxaphene quantitation. 

4.10 Table 4 gives ions that can be used to confirm the presence of organochlorine 

pesticides in the sample. None of these compounds should result in interference with toxaphene 

determination. 

5.0 SAFETY 

This method does not address all safety issues associated with its use. The laboratory is 

responsible for maintaining a safe work environment and a current awareness file of OSHA 

regulations regarding the safe handling of the chemicals specified in this method. A reference 

file of material safety data sheets (MSDSs) should be available to all personnel involved in these 

analyses. 
Method 8276 8276 - 7 Revision 1 

 July 2014 

6.0 EQUIPMENT AND SUPPLIES 

The mention of trade names or commercial products in this manual is for illustrative 

purposes only, and does not constitute an EPA endorsement or exclusive recommendation 

for use. The products and instrument settings cited in SW-846 methods represent those used 

during method development or subsequently evaluated by the Agency. Glassware, reagents, 

supplies, equipment, and settings other than those listed in this manual may be employed 

provided that method performance appropriate for the intended application has been 

demonstrated and documented. 

6.1 Gas chromatograph (GC) - An analytical system equipped with a temperature 

programmable oven suitable for any applicable injection technique and all required accessories 

including syringes, analytical columns, and gases. The capillary column should elute directly into 

the ion source of the mass spectrometer. 

6.2 GC columns - The columns listed in this section were used in developing the 

method and are not intended to exclude others that are available or may be developed. 

Laboratories may use these columns or other columns provided that they document method 

performance data (e.g., chromatographic resolution, analyte breakdown, and sensitivity) 

appropriate for the intended application. 

Given the performance data available to date, the DB-XLB stationary phase columns 

may provide the maximum practical resolving power for congener separations. 

6.2.1 30-m x 0.25-mm ID fused-silica capillary column DB-XLB MSD 

(J&W Scientific), 0.25-μm film thickness 

6.2.2 30-m x 0.25-mm ID fused silica capillary column ZB-MultiResidue-1 

(Phenomenex), 0.25-μm film thickness. This column appears similar to DB-XLB 

MSD. 

 6.2.3 40-m x 0.18-mm ID fused silica capillary column DB-5MS (J&W 

Scientific), 0.18- μm film thickness 

NOTE: DB-5 columns have been problematic, however, in separating some of the 

Parlars, which thus results in summed values. 

6.3 Column rinsing kit - Bonded-phase column rinse kit (J&W Scientific, Catalog No. 

430-3000 or equivalent). 

6.4 Mass spectrometer 

6.4.1 The mass spectrometer must be capable of producing a mass 

spectrum in the negative ion mode under chemical ionization conditions. Before acquiring 

spectra in this mode the instrument must be tuned under NICI conditions according to 

the guidance outlined in Sec. 11.4.1 and using a customized NICI tune table.

6.4.2 An ion trap mass spectrometer may be used if capable of axial 

modulation to reduce ion-molecule reactions and the production of NCI spectra. The 

mass spectrometer must be capable of producing a spectrum using a NCI-customized 

tuning file according to the guidance outlined in Sec. 11.4. 
Method 8276 8276 - 8 Revision 1 

 July 2014 

6.4.3 GC/MS interface - Any GC-to-MS interface may be used that gives 

acceptable calibration points for each compound of interest and achieves acceptable 

tuning performance criteria. For a narrow-bore capillary column, the interface is 

usually capillary-direct into the mass spectrometer source. 

6.5 Data system - A computer system should be interfaced to the mass 

spectrometer. The system must allow the continuous acquisition and storage on machinereadable media of all mass spectra obtained throughout the duration of the chromatographic 

program. The computer should have software that can search any GC/MS data file for ions of a 

specific mass and can plot such ion abundances versus time or scan number. This type of plot 

is defined as an extracted ion current profile (EICP). Software should also be available that 

allows integrating the abundances in any EICP between specified time or scan-number limits. 

6.6 Analytical balance, capable of weighing to 0.0001 g 

7.0 REAGENTS AND STANDARDS 

7.1 Reagent-grade or pesticide-grade chemicals, at a minimum, should be used in 

all tests. Unless otherwise indicated, all reagents should conform to specifications (where 

available) of the Committee on Analytical Reagents of the American Chemical Society. Other 

grades may be used provided the reagent is of sufficiently high purity to permit its use without 

lessening the accuracy of the determination. Reagents should be stored in glass to prevent 

leaching of contaminants from plastic containers. 

NOTE: Store all standard solutions (stock, composite, calibration, internal, and surrogate) at 

≤6°C in polytetrafluoroethylene (PTFE) sealed glass containers in the dark. When a 

standard lot is prepared, aliquots should be stored in individual small vials. All stock 

standards must be replaced after one year or sooner if routine QC (Sec. 9.0) indicates 

a problem. All other standard solutions must be replaced after six months or sooner if 

routine QC (Sec. 9.0) deteriorates. 

7.2 Solvents used in the extraction and cleanup procedures (see appropriate 3500 

and 3600 series methods) may include n-hexane, diethyl ether, methylene chloride 

(dichloromethane), acetone, ethyl acetate, and isooctane (2,2,4-trimethylpentane). All solvents 

should be pesticide grade quality or equivalent, and each lot of solvent should be demonstrated 

to have negligible contamination of both target and non-target compounds (e.g., phthalates) at a 

minimum below the typical laboratory reporting limit. 

7.3 Organic-free reagent water - Reagent water must be interference free. 

All references to water in this method refer to organic-free reagent water unless 

otherwise specified. 

7.4 Stock standard solutions (1000 mg/L) - May be prepared from pure standard 

materials or can be purchased as certified solutions. Other concentrations may be used as 

appropriate for the intended application. If sufficient neat material exists, prepare stock 

standard solutions by accurately weighing 0.0100 g of pure compound. Dissolve the compound 

in isooctane or hexane and dilute to volume in a 10-mL volumetric flask. If compound purity is 

≥96%, the weight may be used without correction to calculate the concentration of the stock 
Method 8276 8276 - 9 Revision 1 

 July 2014 

standard solution. Commercially prepared stock standard solutions may be used at any 

concentration if they are certified by the manufacturer or by an independent source. 

7.5 Composite stock standard - May be prepared from individual stock solutions. 

7.5.1 As an example, for composite stock standards containing <25 

components, add solvent to 1 mL of each individual stock solution at a concentration 

of 1000 mg/L (or other concentrations as appropriate) and mix the solutions in a 25-

mL volumetric flask. The resulting concentration of each component in the mixture, 

after the volume is adjusted to 25 mL, will be 1 mg/25 mL or 40 mg/L. This composite 

solution can be further diluted to obtain the desired concentrations. 

7.5.2 For composite stock standards containing > 25 components, use 

volumetric flasks of the appropriate volume (e.g., 50-mL, 100-mL), and follow the 

procedure described above. 

7.6 Calibration standards - Should be prepared at a minimum of five different 

concentrations by dilution of the composite stock standard with isooctane or hexane and 

should match the sample extract solvent. Recommended standard concentrations for 

establishing a calibration curve for toxaphene are 50 - 400 pg/μL and 1.0 - 500 pg/μL for all 

toxaphene congeners, except Parlar 62. The recommended standard concentrations for 

establishing a calibration curve for Parlar 62 are 5 - 500 pg/μL. Ranges may be extended 

provided that the linear response can be adequately verified through satisfaction of all 

calibration criteria and QC requirements. The low standard must be equivalent to or below the 

lowest result to be reported. All reported results must be within the calibration range. 

Concentrations should correspond to the expected amounts found in real samples and should 

bracket the linear range of the detector. See Method 8000 for additional information on the 

preparation of calibration standards. 

Analysts should evaluate the specific toxaphene standard carefully. Some toxaphene 

components, particularly the more heavily chlorinated, are subject to dechlorination 

reactions. Consequently, standards from different vendors may exhibit marked differences 

which could lead to possible false negatives or to large differences in quantitative results. 

7.7 Internal standard - PCB congener #204 at a concentration range of 50 - 100 

pg/μL in the final extract is suggested for use as an internal standard for the quantitation of 

toxaphene and toxaphene congeners. For example, spike 2 - 4 μL of a 25-ng/μL solution into 

each 1 mL of sample extract for a final concentration of 50 - 100 ng/mL (pg/μL). Other 

concentrations and volumes are acceptable. 

7.8 Surrogate standards - The performance of the method should be monitored 

using surrogate compounds. Surrogate standards are added to all samples, method blanks, 

matrix spikes, and calibration standards. The following compounds are recommended as 

possible surrogates. Other surrogates may be used provided that the analyst can demonstrate 

and document performance appropriate for the data quality needs of the particular application. 

Method 3500 describes the procedures for preparing these surrogates. 

7.8.1 є-hexachlorocyclohexane (є-HCH) and decachlorobiphenyl (PCB 

congener #209) have been found to be a useful pair of surrogates. Decachlorobiphenyl 

should have a minimum retention time of 45 minutes to ensure adequate resolution of 
Method 8276 8276 - 10 Revision 1 

 July 2014 

target compounds. 

7.8.2 Alternatively, if an adequate response can be achieved for the 

particular application, tetrachloro-m-xylene or possibly isotopically labeled toxaphene 

congeners may be used. 

8.0 SAMPLE COLLECTION, PRESERVATION, AND STORAGE 

 Sample collection, preservation, and storage requirements may vary by EPA program 

and may be specified in a regulation or project planning document that requires compliance 

monitoring for a given contaminant. Where such requirements are specified in the regulation, 

follow those requirements. In the absence of specific regulatory requirements, use the following 

information as guidance in determining the sample collection, preservation, and storage 

requirements. 

8.1 See Chapter Four, "Organic Analytes" for sample collection and preservation 

instructions. 

8.2 Store the sample extracts at ≤6 °C, protected from light, in sealed vials (e.g., 

screw- cap or crimp- capped vials) equipped with intact PTFE-lined septa. Extracts should be 

analyzed within 40 days of extraction. 

9.0 QUALITY CONTROL 

9.1 Refer to SW-846 Chapter One for guidance on quality assurance (QA) and QC 

protocols. When inconsistencies exist among QC guidelines, method-specific criteria take 

precedence over both technique-specific and Chapter One criteria. Technique-specific QC 

criteria take precedence over Chapter One criteria. Any effort involving the collection of 

analytical data should include development of a structured and systematic planning 

document, such as a Quality Assurance Project Plan (QAPP), Standard Operating Procedure 

(SOP) or a Sampling and Analysis Plan (SAP); these documents translate project objectives 

and specifications into directions for implementing the project and assessing the results. 

Each laboratory should maintain a formal QC program and maintain records to 

document the quality of the data generated. Development of in-house QC limits for each 

method is encouraged. Use of instrument specific QC limits is encouraged provided such limits 

will generate data appropriate for use in the intended application. All data sheets and QC data 

should be saved for reference or inspection. 

9.2 Refer to Method 8000 for specific determinative method QC procedures and to 

Method 3500 and 5000 for QC procedures to ensure the proper operation of the various sample 

preparation techniques. If an extract cleanup procedure is performed, refer to Method 3600 for 

the appropriate QC procedures. Any more specific QC procedures provided in this method will 

supersede those noted in Methods 8000, 5000, 3500, or 3600. 

9.3 QC procedures necessary to evaluate the GC system operation are found in 

Method 8000 and include evaluation of retention time windows, calibration verification, and 

chromatographic analysis of samples. In addition, discussions regarding the instrument QC 

requirements listed below can be found in the referenced sections of this method. 
Method 8276 8276 - 11 Revision 1 

 July 2014 

9.3.1 The GC/MS is tuned and calibrated using a NICI-customized tune 

table. The analytical system should be tuned prior to the initial calibration and for 

each occurrence when a new initial calibration may be necessary. See Sec. 11.4.1 

for further details.

9.3.2 There must be an initial calibration of the GC/MS system and 

subsequent second source verification as described in Sec.11.4. 

9.3.3 The GC/MS system must meet the calibration verification 

acceptance criteria in Sec. 11.5 every 12 hours. 

9.3.4 The absolute or relative retention time (RRT) of each sample 

component must fall within the specified window of its corresponding standard 

component provided in Sec. 11.4.4. 

9.4 Initial demonstration of proficiency - The initial demonstration of method 

proficiency (IDP) must be performed by the laboratory prior to independently running an 

analytical method, and should be repeated if other changes occur (e.g., instrument repair, 

significant change in procedure, and change in analyst). Refer to Method 8000 Sec. 9.0 for 

additional information regarding instrument, procedure, and analyst IDPs. An IDP must consist 

of replicate reference samples from each sample preparation and determinative method 

combination it utilizes by generating data of acceptable accuracy and precision for target 

analytes in a clean reference matrix taken through the entire preparation and analysis. If an 

autosampler is used to perform sample dilutions, prior to use, the laboratory should satisfy 

itself that those dilutions are of equivalent or better accuracy than is achieved by an 

experienced analyst performing manual dilutions. 

9.5 Method blank - Before processing any samples, the analyst should demonstrate 

that all parts of the equipment in contact with the sample and reagents are interference-free 

through analysis of a method blank. The blank should be carried through all stages of sample 

preparation and analysis. As a continuing check, each time samples are extracted, cleaned up, 

and analyzed, a method blank must be prepared and analyzed for the compounds of interest 

as a safeguard against laboratory contamination. If a peak is observed within the retention time 

window of an analyte that would prevent its determination, the source must be found and 

eliminated, if possible, before samples are processed. Method blank re-extraction may be 

necessary if the source of contamination cannot be determined. 

When new reagents or chemicals are received, the lab should monitor the preparation 

and/or analysis blanks associated with samples for any signs of contamination. It is not 

necessary to test every new batch of reagents or chemicals prior to sample preparation if the 

source shows no prior problems. However, if reagents are changed during a preparation batch, 

separate blanks need to be prepared for each set of reagents. 

 9.6 Sample QC for preparation and analysis - The laboratory must also have 

procedures for documenting matrix effect on method performance (precision, accuracy, 

method sensitivity). Minimally, this should include analysis of QC samples including a 

method blank, a matrix spike/matrix spike duplicate (MS/MSD) or a sample duplicate (if a 

matrix spike duplicate cannot be prepared and sufficient sample exists), and a laboratory 

control sample (LCS) in each analytical batch. Addition of surrogates to each field sample 
Method 8276 8276 - 12 Revision 1 

 July 2014 

and QC sample is also recommended. Method blanks, matrix spike samples, and replicate 

samples should be subjected to the same analytical procedures (Sec. 11.0) as those used 

on actual samples. 

9.6.1 Documenting the effect of the matrix should include analysis of at least 

one matrix spike and one duplicate unspiked sample or one MS/MSD pair. The decision 

on whether to prepare and analyze duplicate samples or a MS/MSD must be based on 

knowledge of the samples in the sample batch. If samples are expected to contain target 

analytes, laboratories may use a matrix spike and a duplicate analysis of an unspiked 

field sample. If samples are not expected to contain target analytes, then laboratories 

should use a MS and MSD. 

9.6.2 An LCS should be included with each analytical batch and consists of 

an aliquot of a clean (control) matrix similar to the sample matrix and of the same 

weight or volume. The LCS is spiked in identical manner as the matrix spike, when 

appropriate. When results of the matrix spike analysis indicate a potential problem due 

to the sample matrix itself, the LCS results are used to verify that the laboratory can 

perform the analysis in a clean matrix. 

9.6.3 Also see Method 8000 for the details on carrying out sample QC 

procedures for preparation and analysis. In-house method performance criteria for 

evaluating method performance should be developed using the guidance found in 

Method 8000. 

9.7 Lower limit of quantitation (LLOQ) check standard 

The laboratory shall establish the LLOQ as the lowest point of quantitation, which in 

most cases, is the lowest concentration in the calibration curve. The LLOQ verification is 

recommended for each project application to validate quantitation capability at low analyte 

concentration levels. This verification may be accomplished with either clean control material 

(e.g., reagent water, solvent blank, Ottawa sand, diatomaceous earth, etc.) or a representative 

sample matrix, free of target compounds. Optimally, the LLOQ should be less than the desired 

regulatory action levels based on the stated DQOs. 

 9.7.1 Determination of LLOQs using spiked clean control material 

represents a best-case scenario, and does not evaluate potential matrix effects of 

real-world samples. For the application of LLOQs on a project-specific basis with 

established DQOs, a representative matrix-specific LLOQ verification may provide 

a more reliable estimate of the lower quantitation limit capabilities. 

9.7.1.1 A LLOQ check standard (not part of an initial calibration) is 

prepared by spiking a clean control material with the analyte(s) of interest at 

the predicted LLOQ concentration level(s). Alternatively, a representative 

sample matrix may be spiked with the analytes of interest at the predicted 

LLOQ concentration levels. The LLOQ check is carried through the same 

preparation procedures as the environmental samples and other QC samples. 

9.7.1.2 Recovery of target analytes in the LLOQ check standard 

should be within established in-house limits, or other such project-specific 

acceptance limits, to demonstrate acceptable method performance at the LLOQ. 

Until the laboratory has sufficient data to determine acceptance limits, the LCS 
Method 8276 8276 - 13 Revision 1 

 July 2014 

criteria ± 20% may be used for the LLOQ acceptance criteria. This acknowledges 

the poorer overall response at the low end of the calibration curve. Historicallybased LLOQ acceptance criteria should be determined as soon as practical once 

sufficient data points have been acquired. 

9.7.2 In-house acceptance criteria for recovery of the LLOQ check standard 

for a sample matrix can be calculated when sufficient data points exist. The laboratory 

should have a documented procedure for establishing in-house acceptance ranges. 

9.8 Surrogate recoveries - The laboratory should evaluate recovery data from 

individual samples against historically developed surrogate control limits. See Method 

8000 for information on evaluating surrogate data and developing and updating surrogate 

limits. Procedures for evaluating recoveries of multiple surrogates and associated 

corrective actions when they are deemed unacceptable should be defined in a QAPP or 

SOP. 

9.9 The experience of the analyst performing GC-NICI/MS is invaluable to the 

success of the method. Each day that analysis is performed, the calibration verification 

standard should be evaluated to determine if the chromatographic system is operating properly. 

Do the peaks look normal? Is the response obtained comparable to the response from previous 

calibrations? 

Careful examination of the standard chromatogram can indicate if the column is still 

performing acceptably, the injector is leaking, the injector septum needs replacing, etc. When 

any changes are made to the system (e.g., the column or septum is changed), see guidance 

in Method 8000 regarding whether recalibration of the system must take place. 

9.10 It is recommended that the laboratory adopt additional QA practices for use 

with this method. Specific practices that are most productive depend upon the needs of the 

laboratory, the nature of the samples, and project-specific requirements. Field duplicates 

may be analyzed to assess precision of the environmental measurements. When doubt 

exists over identification of a peak on the chromatogram, confirmatory techniques such as 

GC with a dissimilar column, element-specific detector, or mass spectrometer (selected ion 

monitoring or full scan) must be used. Whenever possible, the laboratory should analyze 

standard reference materials and participate in relevant performance evaluation studies. 

10.0 CALIBRATION AND STANDARDIZATION 

See Sec. 11.4 and 11.5 for information on calibration and standardization. 

 
Method 8276 8276 - 14 Revision 1 

 July 2014 

11.0 PROCEDURE 

11.1 Sample preparation 

Samples are normally prepared by one of the following methods prior to GC-NICI/MS 

analysis. 

Matrix Methods 

Water 3510, 3520 

Soil/sediment 3540, 3541, 3545, 3546, 3550, 3562 

Tissue 3540, 3541, 3545, 3550, 3562 

11.2 Extract cleanup - Cleanup procedures may not be necessary for a relatively 

clean sample matrix, but most extracts from environmental and waste samples will require 

additional preparation before analysis. The specific cleanup procedure used will depend on the 

nature of the sample to be analyzed, expected interferences coextracted from the sample, and 

the DQOs. General guidance on the selection of cleanup methods is provided in Sec. 11.2 

and in Method 3600. Refer to Table 13 for a data comparison of real-world samples extracted 

with and without cleanup procedures. 

11.2.1 Method 3610 (alumina) may be used to remove phthalate esters. 

11.2.2 Method 3620 (Florisil®) may be used to separate organochlorine 

pesticides from aliphatic compounds, aromatics, and nitrogen-containing compounds. 

11.2.3 Method 3630 (silica gel) may be used to separate single- and 

multi- component organochlorine compounds from interferences. 

11.2.4 Method 3640 (GPC - pesticide option) is a size exclusion 

procedure that may be used to remove high molecular weight materials. 

11.2.5 Method 3660 (sulfur cleanup) should be used to remove possible 

sulfur interfering compounds which may be present in certain sediments and industrial 

wastes. (These compounds should not interfere with monitored ions from toxaphene 

but should nevertheless be removed because of deleterious effects on 

chromatography and on the response of the NICI/MS ion source during coelution.) 

11.2.6 Method 3665 (sulfuric acid/permanganate) may be used prior to 

toxaphene analysis to remove more fragile organic contaminants. 

11.3 GC conditions - This method uses a single column in conjunction with an 

electron capture NICI/MS detector. If an autosampler is not used, the use of an internal 

standard is required. It is highly recommended to use an internal standard in all samples even 

if an autosampler is used. The list in Sec. 6.0 indicates the columns used to develop the 

method performance data in the tables at the end of this method and is not intended to exclude 

other columns that are available or may be developed. Laboratories may use these or other 

capillary columns (including columns of other dimensions), provided that they document 

method performance data (e.g., chromatographic resolution, analyte breakdown, and 

sensitivity) appropriate for the intended application. 
Method 8276 8276 - 15 Revision 1 

 July 2014 

11.4 Tuning, initial calibration, and second-source calibration accuracy check - 

Establish the GC-NICI/MS operating conditions according to the recommendations in Table 

1. Alternative operating conditions may be acceptable provided that appropriate compound 

sensitivity and selectivity can be achieved. 

11.4.1 Prior to analysis of samples or calibration standards, the instrument 

must be tuned for NCI conditions. If possible, use the CI autotune procedure 

provided by the instrument manufacturer. Without specific manufacturer guidance, 

the following generic recommendations should be followed. 

11.4.1.1 The source is operated in the CI mode using methane as 

the reagent gas. 

11.4.1.2 When the source pressure has stabilized at a value known 

to produce satisfactory results, the instrument is manually tuned and calibrated 

using a NCI-customized tuning file. Pressure stabilization usually takes about 

45 minutes. 

NOTE: The internal standard noted in Sec. 7.7 should permit most of the 

components of interest in a chromatogram to have retention times 

of 0.80-1.20 relative to it. Use the base peak ion from the internal 

standard as the primary ion for quantitation (Table 2). 

11.4.2 Analyze 1 μL of each calibration standard (containing the quantitation 

compounds, as well as, the appropriate surrogates and internal standard) and calculate 

the response factor (RF) as shown in Sec. 11.4.3. A set of at least five calibration 

standards is necessary (Sec. 7.6 and Method 8000). Alternate injection volumes may 

be used if applicable QC requirements are met, but must be the same for all standards 

and sample extracts. Figure 1 is a representative chromatogram of select congeners. 

NOTE: Toxaphene will require a separate calibration curve from the congeners due 

to possible congener interferences present in the toxaphene calibration 

standard chromatograms. 

11.4.3 Initial calibration calculations 

11.4.3.1 Calculate a RF for each target analyte relative to one of 

the internal standards as follows: 

= 	

			

				

		

where: 

As = Peak response of the analyte or surrogate. 

Ais = Peak response of the internal standard. 

Cs = Concentration or mass of the analyte or surrogate in the sample aliquot. 

Cis= Concentration or mass of the internal standard in the sample aliquot.
Method 8276 8276 - 16 Revision 1 

 July 2014 

11.4.3.2 Calculate the average RF and the percent relative 

standard deviation (%RSD) of the RFs for each target analyte using the 

equations below. The %RSD should be ≤20% for each target analyte. It is also 

recommended that a minimum RF for the most common target analytes be 

demonstrated for each individual calibration level to ensure that these 

compounds are behaving as expected. In addition, meeting the minimum RF 

criteria for the lowest calibration standard is critical in establishing and 

demonstrating the desired sensitivity. 

Due to the large number of compounds that may potentially be 

analyzed by this method, some compounds will fail to meet these criteria. For 

these occasions, it is acknowledged that the failing compounds may not be 

critical to the specific project and therefore they may be used as qualified data or 

estimated values for screening purposes. The analyst should strive to place 

more emphasis on meeting the calibration criteria for compounds that are critical 

to the project. 



	= 	 	  = 	

∑ 







 = 

∑ 

 





 = 	





		100

where: 

	= RF for each of the calibration standards. 

	 = Average RF for each compound from the initial calibration. 

n = Number of calibration standards (e.g., 5). 

11.4.3.3 If more than 10% of the compounds included in the initial 

calibration exceed the 20% RSD limit or do not meet the minimum coefficient of 

determination (r2 = 0.99) for alternate curve fits, the chromatographic system 

may be considered too reactive for analysis to begin. Clean or replace the 

injector liner, capillary column, and/or ion source, and then repeat the calibration 

procedure beginning with Sec. 11.4. 

Either of the two procedures described in Sec. 11.4.4.1 or 11.4.4.2 

may be used to determine calibration function acceptability. These include 

refitting the calibration data back to the model or the determination of the 

relative standard error (RSE) for the curve when comparing the actual response 

with the predicted response. 

11.4.4.1 Refitting the calibration data back to the model 

or calculating the % Error is determined by using the following 

equation: 

%	#$ = %&'%

(

&

%&

		100	
Method 8276 8276 - 17 Revision 1 

 July 2014 

where:

x’i = Measured amount of analyte at calibration level i, in mass or 

concentration units. 

xi = True amount of analyte at calibration level i, in mass or 

concentration units. 

Percent error between the calculated and expected amounts of 

an analyte should be ≤30% for all standards. For some data uses, 

≤50% may be acceptable for the lowest calibration point. 

11.4.4.2 Relative Standard Error is calculated by using the 

following equation:

 

# = 100 × -. /	

0 − 	

	

2

 3

	4

9(6 − 7)

where: 

xi = True amount of analyte in calibration level i, in mass or 

concentration units. 

x ́i = Measured amount of analyte in calibration level i, in mass or 

concentration units. 

p = Number of terms in the fitting equation. 

 (average = 1, linear = 2, quadratic = 3, cubic = 4)

n = Number of calibration points. 

RSE acceptance limit criterion for the calibration model is ≤20%.

11.4.5 Evaluation of retention times -Either the absolute or relative retention 

time (RRT) used in conjunction with spectral matching is allowed for use by the 

laboratory to establish peak identity. The absolute or RRT of each target analyte in 

each calibration standard should agree within established limits set by the laboratory 

in their SOP. These limits should be appropriate for reliable identification of the 

target analyte. If this criterion is not met and there are no other indicators of an 

analyte's identification (such as a very unique and high probability mass spectral 

match), that analyte may not be considered as identified using either RRT or 

absolute retention times. The RRT equation follows units. 

= = 	

>6>?$6	>?@	$A	>h	6
CD>

>6>?$6	>?@	$A	>h	E6>6
C	>
6F
F
Method 8276 8276 - 18 Revision 1 

 July 2014 

11.4.6 Linearity of target analytes - If the RSD of any target analyte is ≤20%, 

then the relative response is assumed to be constant over the calibration range, and 

the average RF may be used for quantitation (Sec. 11.8.2). 

11.4.6.1 If the RSD of any target analyte is >20%, refer to Method 

8000 for additional calibration options. One of the options must be applied to the 

GC/MS calibration in this situation, or a new initial calibration must be performed. 

The average RF should not be used for compounds that have an RSD >20% 

unless the concentration is reported as estimated. 

11.4.6.2 When the RSD is >20%, plotting and visual inspection of a 

calibration curve can be a useful diagnostic tool. The inspection may indicate 

analytical problems, including errors in standard preparation, presence of active 

sites in the chromatographic system, analytes that exhibit poor chromatographic 

behavior, etc. 

11.4.6.3 Due to the large number of compounds that may be 

analyzed by this method, some compounds may fail to meet either the 20% 

RSD, minimum coefficient of determination (r2 = 0.99), or the acceptance criteria 

for alternative calibration procedures in Method 8000. Any calibration method 

described in Method 8000 may be used for a compound, but it should be used 

consistently until the next calibration is performed. If compounds fail to meet 

these criteria, the associated concentrations may still be determined but they 

must be reported as estimated. In order to report non-detects, it must be 

demonstrated that there is adequate sensitivity to detect the failed compounds 

at the applicable lower quantitation limit. 

11.4.6.4 The method of linear regression analysis has the potential 

for a significant bias at the lower portion of a calibration curve, while the 

average response factor and quadratic methods of calibration do not typically 

have this problem. Also, when a least squares linear regression calibration 

model is used, inverse weighting of the concentrations used in the initial 

calibration has been shown to emphasize precision at the low end of the 

calibration range (see Method 8000 for all calibration model options). 

When evaluating the calibration curves with the linear regression model, 

the analyst should perform a minimum quantitation check on the viability of the 

lowest calibration point by refitting the response from the low concentration 

calibration standard back into the curve (see Method 8000 for additional details). 

It is not necessary to reanalyze a low concentration standard; rather the data 

system can recalculate this low concentration as if it were an unknown sample. 

The recalculated concentration of the low calibration point should be ±50% (or inhouse historically based acceptance criteria) of the standard’s true concentration. 

Other recovery criteria may be applicable depending on the project’s 

DQOs. For those situations, the minimum quantitation check criteria should be 

outlined in a laboratory SOP, or a project-specific QAPP. Analytes which do not 

meet the minimum quantitation calibration refitting criteria should be considered 

"out-of-control." Corrective actions may be appropriate such as redefining the 

LLOQ and/or reporting "out-of-control" target analytes as estimated when the 
Method 8276 8276 - 19 Revision 1 

 July 2014 

concentration is at or near the lowest calibration point. 

11.4.7 The initial calibration function for each target analyte should be 

checked by injecting the second-source standard immediately after the standards 

used for initial calibration. The second-source standard should be prepared at a 

concentration near the middle of the calibration range with a standard from a source 

different (if available) from that used for the initial calibration. The measured values 

of the parameters in the second-source check standard should fall within 70 - 130% 

of the expected value(s). Due to the variability of technical mixtures (e.g., technical 

toxaphene), this criterion for the second source check standard should be applied 

with caution. An alternative recovery limit may be appropriate based on the desired 

project-specific DQOs. Quantitative sample analyses should not proceed for those 

analytes that fail the second-source standard initial calibration verification. 

However, analyses may continue for those analytes that fail the criteria with an 

understanding these results could be used for screening purposes and would be 

considered estimated values. 

11.5 Calibration verification consists of several steps that are performed at 

the beginning of each 12-hour analytical shift. 

11.5.1 Calibration verification - Initial calibration (Sec. 11.4) for each compound 

of interest should be verified once every 12 hours prior to sample analysis, with the 

introduction technique and conditions used for samples. This is accomplished by 

analyzing a calibration verification standard (containing all the compounds for 

quantitation) at a concentration either near the midpoint concentration for the calibration 

range of the GC-NICI/MS or near the action level for the project. The results must be 

compared against the most recent initial calibration curve and should meet the 

verification acceptance criteria provided in Sec. 11.5.1.1 through 11.5.1.5. 

11.5.1.1 Each of the target analytes in the calibration verification 

standard should meet the appropriate minimum RFs. These criteria are 

particularly important when the target analytes are also critical projectrequired compounds. This is the same check that is applied during the initial 

calibration. 

11.5.1.2 If minimum RFs are not met, the system should be 

evaluated, and corrective action taken before sample analysis begins. 

Possible problems include standard mixture degradation; contamination of the 

injection port inlet, source and/or front end of the column; active sites in the 

column or chromatographic system; etc. 

11.5.1.3 All target compounds of interest must be evaluated using a 

20% criterion: percent difference when performing the average RF model 

calibration and percent drift when calibrating with a regression fit model. Refer to 

Method 8000 for guidance on calculating percent difference and drift. 

11.5.1.4 If the percent difference or percent drift for a compound is 

≤20%, the initial calibration for that compound is assumed to be valid. Due to 

large numbers of compounds that may potentially be analyzed by this method, it

is expected that some compounds will fail to meet the criterion. If the criterion is 
Method 8276 8276 - 20 Revision 1 

 July 2014 

not met for more than 20% of the compounds included in the initial calibration, 

then corrective action must be taken prior to the analysis of samples. When 

compounds fail, they may still be reported as non-detects if it can be 

demonstrated that there was adequate sensitivity to detect the compound at the 

applicable quantitation limit. For situations when the failed compound is present, 

the concentrations in the affected samples must be reported as estimated. 

11.5.1.5 Problems similar to those listed under initial calibration could 

affect the ability to pass the calibration verification standard analysis. If the 

problem cannot be corrected by other measures, a new initial calibration must be 

generated. Calibration verification criteria must be met before sample analysis 

may begin. 

11.5.2 Method blank - A method blank should be analyzed prior to sample 

analysis to demonstrate that the total system (introduction device, transfer lines and 

GC-NICI/MS system) is free of contaminants. If the method blank indicates 

contamination, it may be appropriate to analyze a solvent blank to demonstrate that 

the contamination is not a result of carryover from standards or samples. See Method 

8000 for information regarding method blank performance criteria. 

11.5.3 Internal standard retention time - Retention times of the internal 

standard in the calibration verification standard must be evaluated immediately 

during or after data acquisition. If the retention time for any internal standard 

changes by >30 seconds from that in the midpoint standard level of the most recent 

initial calibration sequence, the chromatographic system must be inspected for 

malfunctions and corrections must be made, as required. When corrections are 

made, reanalysis of samples analyzed while the system was malfunctioning is 

required. 

11.5.4 Internal standard response - If the EICP area for the internal standard in 

the calibration verification standard changes by greater than a factor of two (-50% to 

+100%) from that in the midpoint standard level of the most recent initial calibration 

sequence, the mass spectrometer must be inspected for malfunctions and corrections 

must be made, as appropriate. When corrections are made, reanalysis of samples 

analyzed while the system was malfunctioning is required. 

NOTE: A decrease in the EICP area of the internal standard may be due to the use 

of a hydrocarbon reagent gas (e.g., methane) in the negative ion chemical 

ionization mode and a decreased source temperature (160 °C, versus 250 

°C for EI). These conditions make the ion source susceptible to a buildup of 

a hydrocarbon film on its surface. This in turn may lead to a lower signal. If 

this decrease of signal is gradual, it should not signify anything worse than 

this hydrocarbon buildup. If the signal drops drastically over one or two runs, 

something else is happening and measures should be taken immediately to 

remedy this. 

11.6 GC-NICI/MS analysis of samples 

11.6.1 It is highly recommended that sample extracts be screened on a GC with 

an electron capture detector (ECD) using the same type of capillary column employed 

in the GC-NICI/MS system. Pre-screening can detect the possible presence of 
Method 8276 8276 - 21 Revision 1 

 July 2014 

interferences (e.g., sulfur, PCBs) to determine if extract cleanups are needed. 

Screening will minimize contamination of the GC-NICI/MS system from unexpectedly 

high concentrations of organic compounds. The QAPP often contains historical 

contaminant information of the sampling site which may indicate if screening is 

necessary. 

11.6.2 Allow the sample extract to warm to room temperature. Just prior to 

analysis, add an aliquot of the internal standard solution to the concentrated sample 

extract. The internal standard concentration in the injected sample extract must be 

the same as that in the calibration standards. 

11.6.3 Inject an aliquot of the sample extract using the same operating 

conditions employed for the calibration (Sec. 11.4). The volume to be injected should 

include an appropriate concentration that is within the calibration range of the 

standards as noted in Sec. 7.7. The injection volume must be the same volume used 

for the calibration standards. 

11.6.4 If any quantitation ion response exceeds the GC-NICI/MS system 

initial calibration range, the sample extract must be diluted and reanalyzed. Additional 

internal standard solution must be added to the diluted extract to maintain the same 

concentration as in the calibration standards (usually 50 - 100 pg/μL or other 

concentrations as appropriate). 

NOTE: It may be a useful diagnostic tool to monitor internal standard retention times 

in all samples, spikes, blanks, and standards to check drifting, method 

performance, poor injection execution, and the need for system inspection 

and/or maintenance. Internal standard responses (area counts) must be 

monitored in all samples, spikes, blanks for similar reasons. If the EICP area 

for the internal standard in samples, spikes and blanks changes by a factor of 

two (-50% to +100%) from the areas determined in the calibration verification 

standard analyzed that day, corrective action must be taken. The samples, 

spikes, or blanks should be reanalyzed or the data qualified. 

11.6.4.1 When ions from a compound in the sample saturate 

the detector, this analysis should be followed by an instrument blank 

consisting of clean solvent. If the blank analysis is not free of interferences, 

then the system must be decontaminated. Sample analysis may not resume 

until the blank analysis is demonstrated to be free of interferences. 

Contamination from one sample to the next on the instrument usually takes 

place in the syringe. If adequate syringe washes are employed, carryover 

from high concentration samples can usually be avoided. 

11.6.4.2 All dilutions should keep the response of the major 

constituents (previously saturated peaks) within the linear range of the 

curve. 

11.6.5 The use of selected ion monitoring (SIM) is preferred. Using the primary 

ion for quantitation and the secondary ions for confirmation sets up the collection 

groups based on their retention times. Most compounds have a small mass defect in 

their spectra, usually <0.2 amu. These mass defects should be accounted for during 
Method 8276 8276 - 22 Revision 1 

 July 2014 

data acquisition. The total scan time should be <1 second and produce at least 5 -10 

scans per chromatographic peak. The start and stop times for the SIM groups are 

determined from a full scan analysis. 

11.7 Analyte identification

11.7.1 The qualitative identification of compounds determined by this method 

is based on retention time and on comparison of the sample mass spectrum, after 

background correction, with characteristic ions in a reference mass spectrum. The 

reference mass spectrum must be generated by the laboratory using the conditions of 

this method. The characteristic ions from the reference mass spectrum are defined as 

the three ions of greatest relative intensity, or any ions over 30% relative intensity, if 

fewer than three such ions occur in the reference spectrum. Compounds are identified 

when the following criteria are met. 

11.7.1.1 The intensities of the characteristic ions of a compound 

must maximize in the same scan or within one scan of each other. Selection of 

a peak by a data system target compound search routine where the search is 

based on the presence of a target chromatographic peak containing ions 

specific for the target compound at a compound-specific retention time will be 

accepted as meeting this criterion. 

11.7.1.2 Either absolute or RRT, used in conjunction with spectral 

matching is allowed for use by the laboratory to establish peak identity. The 

absolute or RRT of each target analyte, when compared against the daily 

calibration verification standard, should agree within established limits set by 

the laboratory in their SOP. These limits should be appropriate for reliable 

identification of the target analyte. If this criterion is not met and there are no 

other indicators of an analyte's identification (such as a very unique and high 

probability mass spectral match), that analyte may not be considered as 

identified using either RRT or absolute retention times. 

 11.7.1.3 The relative intensities of the characteristic ions agree 

within 30% of the relative intensities of these ions in the reference spectrum. 

(e.g., for an ion with an abundance of 50% in the reference spectrum, the 

corresponding abundance in a sample spectrum should be 20 - 80%.) Use 

professional judgment when interferences are observed. 

11.7.1.4 Structural isomers that produce very similar mass spectra 

should be identified as individual isomers if they have sufficiently different GC 

retention times. Sufficient GC resolution is achieved if the height of the valley 

between two isomer peaks is less than 50% of the average of the two peak 

heights. Otherwise, structural isomers are identified as isomeric pairs. The 

resolution should be verified on the midpoint concentration of the initial 

calibration as well as the laboratory-designated calibration verification standard 

level if closely eluting isomers are to be reported. 

CAUTION: Because of the complexity of toxaphene, decreasing run time will 

likely cause additional isomers to be reported as the target 

congeners, thus inflating concentrations 

 
Method 8276 8276 - 23 Revision 1 

 July 2014 

11.7.1.5 Identification is hampered when sample components are 

not resolved chromatographically and produce mass spectra containing ions 

contributed by more than one analyte. When GC peaks obviously represent 

more than one sample component (i.e., a broadened peak with shoulder(s) or a 

valley between two or more maxima), appropriate selection of analyte spectra 

and background spectra is important. 

11.7.1.6 Examination of EICPs of appropriate ions can aid in the 

selection of spectra and in qualitative identification of compounds. When 

analytes coelute (i.e., only one chromatographic peak is apparent), the 

identification criteria may be met, but each analyte spectrum will contain 

extraneous ions contributed by the coeluting compound. 

11.7.2 The identification of mixtures such as toxaphene is not based on a 

single peak, but rather on the characteristic peaks that comprise the "fingerprint" of the 

mixture, using both the retention times and shapes of the indicator peaks. See Method 

8000 for information on confirmation of tentative identifications. 

11.8 Quantitation 

11.8.1 Multi-component analytes such as toxaphene - Complex analytes 

present problems in measurement. Quantitation is based on the areas of the 

characteristic peaks as compared to the areas of the corresponding peaks at the same 

retention times in the calibration standard, using either internal or external calibration 

procedures. Suggestions are offered in the following sections for handling toxaphene. 

11.8.1.1 Calibrate the instrument for toxaphene using the 

guidance noted in Sec. 11.4 

11.8.1.2 Quantitate toxaphene by summing the area of all peaks 

originating from this compound using the total area of the toxaphene pattern 

or using the 4 to 6 major peaks that closely match the corresponding peaks 

in the most recent calibration verification standard. Whichever approach is 

employed should be documented and available to the data user, if 

necessary. 

11.8.1.3 While toxaphene contains a large number of compounds 

that will produce well-resolved peaks in a GC-NICI/MS chromatogram, it also 

contains many other components that are not chromatographically resolved. 

This unresolved complex mixture results in the "hump" in the chromatogram 

that is characteristic of this compound. Although the resolved peaks are 

important for identification, the area of the unresolved complex mixture 

contributes a significant portion of the area of the total response. 

11.8.1.4 To measure total area, construct the baseline of toxaphene 

in the sample chromatogram between the retention times of the first and last 

eluting toxaphene components in the standard. In order to use the total area 

approach, the pattern in the sample chromatogram must be compared to that of 

the standard to ensure that all of the major components in the standard are 

present in the sample. Otherwise, the sample concentration may be 

significantly underestimated. As an example, either manually or by using the 
Method 8276 8276 - 24 Revision 1 

 July 2014 

data system’s control settings, integrate the entire area under the total ion 

chromatogram for the toxaphene response and subtract out the internal 

standard response (m/z 429.8) and ions associated with surrogate or other

compounds if they are present. 

 11.8.1.5 When toxaphene is determined using the 4 to 6 peaks 

approach, the analyst must take care to evaluate the relative areas of the peaks 

chosen in the sample and standard chromatograms. It is highly unlikely that the 

peaks will match exactly, but the analyst should not employ peaks from the 

sample chromatogram whose relative sizes or areas appear to be 

disproportionally larger or smaller in the sample compared to the standard. 

 11.8.1.6 The heights or areas of the selected 4 - 6 peaks should be 

summed together and used to determine the toxaphene concentration. 

Alternatively, utilize each peak in the standard to calculate a calibration factor for 

that peak, using the total mass of toxaphene in the standard. These calibration 

factors are then used to calculate the concentration of each corresponding peak 

in the sample chromatogram and the 4 - 6 resulting concentrations are averaged 

to provide the final result for the sample. 

11.8.2 Once a target compound has been identified, the quantitation of that 

compound will be based on the integrated abundance of the primary characteristic 

ion from the EICP. 

11.8.2.1 Use the integration produced by the software if the 

integration is correct because the software should produce more consistent 

integrations. However, manual integrations may be necessary when the software 

does not produce proper integrations because baseline selection is improper, the 

correct peak is missed, a coelution is integrated, the peak is partially integrated, 

etc. The analyst is responsible for ensuring that the integration is correct whether 

performed by the software or done manually. 

11.8.2.2 Manual integrations should not be substituted for proper 

maintenance of the instrument or setup of the method (e.g., retention time 

updates, integration parameter files, etc). The analyst should seek to minimize 

manual integration by properly maintaining the instrument, updating retention 

times, and configuring peak integration parameters.

11.8.3 If the RSD of a compound's response factor is ≤20%, the concentration 

in the extract may be determined using the average RF from initial calibration data 

(Sec. 11.4.4). See Method 8000 for the equations describing internal standard 

calibration and either linear or non-linear calibrations. 

11.8.4 Table 5 lists example retention times for the target analytes. The 

retention times listed in this table are provided for illustrative purposes only. Each 

laboratory must determine retention times and retention time windows for their 

specific application of the method. 

 
Method 8276 8276 - 25 Revision 1 

 July 2014 

12.0 DATA ANALYSIS AND CALCULATIONS 

See Secs. 11.4 through 11.8 and Method 8000 for information on data analysis 

and calculations. 

13.0 METHOD PERFORMANCE 

13.1 Performance data and related information are provided in SW-846 methods 

only as examples and guidance. The data do not represent required performance criteria for 

users of the methods. Instead, performance criteria should be developed on a project-specific 

basis, and the laboratory should establish in-house QC performance criteria for the application 

of this method. Performance data must not be used as absolute QC acceptance criteria for the 

purposes of laboratory QC or accreditation. All performance data included resulted from both 

single and multi-laboratory analyses. 

13.2 The chromatographic separations in this method were tested in multiple 

laboratories by using clean hexane and liquid/solid extracts spiked with the test compounds at 

various concentrations. Single-operator and multi-laboratory precision and method accuracy 

were found to be related to the concentration of the compound and the type of matrix. 

13.3 The levels of accuracy and precision that can be achieved with this 

method depend on the sample matrix, sample preparation, optional cleanup techniques, 

and calibration procedures used. 

13.4 Tables 2 through 4 represent SIM for method analytes using GC-NICI/MS. 

13.5 Table 5 contains representative retention times. 

NOTE: Decachlorobiphenyl should have a minimum RT of 45 minutes to ensure 

adequate resolution of target compounds. 

13.6 Table 6 contains single-laboratory performance data on spiked soil for 

toxaphene and chlordane. Data are provided for guidance purposes only. 

13.7 Table 7 contains single-laboratory performance data on spiked soil for 

toxaphene congeners. Data are provided for guidance purposes only. 

13.8 Table 8 contains single-laboratory performance data on spiked 

soil for organochlorine pesticides. Data are provided for guidance purposes only. 

13.9 Table 9 contains multi-laboratory phase I validation data using unknown 

standard concentrations. 

13.10 Table 10 contains multi-laboratory phase II validation data using spiked 

extracts of uncontaminated real-world sample matrices. 

13.11 Table 11 contains multi-laboratory phase II validation data using realworld samples from Terry Creek in Brunswick, Georgia. 

13.12 Table 12 contains multi-laboratory phase II validation data using real world 
Method 8276 8276 - 26 Revision 1 

 July 2014 

samples from Terry Creek in Brunswick, Georgia. Extracts from this study were further 

subjected to copper, acid, and silica gel cleanup methods. 

13.13 Table 13 includes a Terry Creek real-world sample data comparison 

between extraction only to those extracts subjected to copper, acid, and silica gel cleanup 

methods. 

13.14 Table 14 contains multiple-laboratory validation data using multiple 

technical toxaphene spiking protocols of uncontaminated real world sample matrices. 

13.15 Table 15 contains multi-laboratory phase III validation data using spiked 

extracts of uncontaminated real world sample matrices. 

13.16 Table 16 contains multi-laboratory phase III validation data using real 

world samples from Terry Creek in Brunswick, Georgia. 

13.17 Figure 1 is an example chromatogram of a mixed toxaphene congeners 

standard. 

13.18 Figure 2 is an example chromatogram of a fish tissue extract containing 

weathered toxaphene and spiked with decachlorobiphenyl. 

13.19 Figure 3 is an example chromatogram of a toxaphene congener 

validation standard. 

13.20 Figure 4 is an example chromatogram of an EPA Region 4 sediment 

extract spiked with decachlorobiphenyl. 

NOTE: Not all components shown in the figures may be target analytes. 

14.0 POLLUTION PREVENTION 

14.1 Pollution prevention encompasses any technique that reduces or eliminates 

the quantity and/or toxicity of waste at the point of generation. Numerous opportunities for 

pollution prevention exist in laboratory operations. The EPA has established a preferred 

hierarchy of environmental management techniques that places pollution prevention as the 

management option of first choice. Whenever feasible, laboratory personnel should use 

pollution prevention techniques to address their waste generation. When wastes cannot be 

feasibly reduced at the source, the Agency recommends recycling as the next best option. 

14.2 For information about pollution prevention that may be applicable to 

laboratories and research institutions consult Less is Better: Laboratory Chemical management 

for Waste Reduction, a free publication available from the American Chemical Society (ACS), 

Committee on Chemical Safety, 

http://portal.acs.org/portal/fileFetch/C/WPCP_012290/pdf/WPCP_012290.pdf. 
Method 8276 8276 - 27 Revision 1 

 July 2014 

15.0 WASTE MANAGEMENT 

The EPA requires that laboratory waste management practices be conducted consistent 

with all applicable rules and regulations. Laboratories are urged to protect air, water, and land 

by minimizing and controlling all releases from hoods and bench operations, complying with the 

letter and spirit of any sewer discharge permits and regulations, and by complying with all solid 

and hazardous waste regulations, particularly the hazardous waste identification rules and land 

disposal restrictions. For further information on waste management, consult The Waste 

Management Manual for Laboratory Personnel available from the American Chemical Society at 

the address listed in Sec. 14.2. 

16.0 REFERENCES 

1. A.G. Harrison (2nd ed.), Chemical Ionization Mass Spectrometry, CRC Press, Boca Raton, 

FL, 1992. 

2. W. C. Brumley, C. M. Brownrigg, and A. H. Grange, "Determination of Toxaphene in Soil 

by Electron-capture Negative Ion Mass Spectrometry after Fractionation by Highperformance Gel Permeation Chromatography," J. Chromatogr., 633, 177-183, 1993. 

3. Brumley, W. C. “Analytical Protocol (GC/NIMS) For OSW's Response to OIG Report 

(2005-P-00022) on Toxaphene Analysis.” EPA/600/R-08/048. (TIP # 08-069, Published 

Report) U.S. Environmental Protection Agency, Washington, DC, 2008. 

4. V. Lopez-Avila, E. Baldin, J. Benedicto, J. Milanes, W. F. Beckert, "Application of OpenTubular Columns to SW-846 GC Methods," Report for the U.S. Environmental Protection 

Agency, Contract 68-03-3511, Mid-Pacific Environmental Laboratory, Mountain View, CA, 

1990. 

5. "Development and Application of Test Procedures for Specific Organic Toxic Substances 

in Wastewaters," Category 10, Pesticides and PCB Report for the U.S. Environmental 

Protection Agency, Contract 68-03-2606. 

6. D. F. Goerlitz, L. M. Law, "Removal of Elemental Sulfur Interferences from Sediment 

Extracts for Pesticide Analysis," Bull. Environ. Contam. Toxicol., 6, 9, 1971. 

7. S. Jensen, L. Renberg, L. Reutergardth, "Residue Analysis of Sediment and Sewage 

Sludge for Organochlorines in the Presence of Elemental Sulfur," Anal. Chem., 49, 316-

318, 1977. 

8. R. H. Wise, D. F. Bishop, R. T. Williams, B. M. Austern, "Gel Permeation Chromatography 

in the GC/MS Analysis of Organics in Sludges," U.S. Environmental Protection Agency, 

Cincinnati, OH. 

9. H. B. Pionke, G. Chesters, D.E. Armstrong, "Extraction of Chlorinated Hydrocarbon 

Insecticides from Soil," Agron. J., 60, 289, 1968. 

10. J. A. Burke, P. A. Mills, D.C. Bostwick, "Experiments with Evaporation of Solutions of 

Chlorinated Pesticides," J. Assoc. Off. Anal. Chem., 49, 999, 1966. 
Method 8276 8276 - 28 Revision 1 

 July 2014 

11. J. A. Glazer, et al., "Trace Analyses for Wastewaters," Environ. Sci. and Technol., 15, 

1426, 1981. 

12. P. J. Marsden, "Performance Data for SW-846 Methods 8270, 8081, and 8141," U.S.

 Environmental Protection Agency, EMSL-Las Vegas, EPA/600/4-90/015. 

13. V. Lopez-Avila (Beckert, W., Project Officer), "Development of a Soxtec Extraction 

Procedure for Extracting Organic Compounds from Soils and Sediments," EPA 600/X91/140, US Environmental Protection Agency, Environmental Monitoring Systems 

Laboratory, Las Vegas, NV, October 1991. 

14. C. Markell, "Solid-Phase Extraction of TCLP Leachates," Proceedings of the Tenth Annual 

Waste Testing and Quality Assurance Symposium, Arlington, VA, July, 1994. 

15. D. Bennett, B. Lesnik, S. M. Lee, "Supercritical Fluid Extraction of Organochlorine 

Pesticide Residues from Soils," Proceedings of the Tenth Annual Waste Testing and 

Quality Assurance Symposium, Arlington, VA, July, 1994. 

16. C. Markell, "3M Data Submission to EPA," letter to B. Lesnik, June 27, 1995. 

17. B. Richter, J. Ezzell, and D. Felix, "Single Laboratory Method Validation Report - 

Extraction of Organophosphorus Pesticides, Herbicides and Polychlorinated Biphenyls 

Using Accelerated Solvent Extraction (ASE) with Analytical Validation by GC/NPD and 

GC/ECD," Dionex, Salt Lake City, UT, Document 101124, December 2, 1994. 

18. K. Li, J. M. R. Bélanger, M. P. Llompart, R. D. Turpin, R. Singhvi, and J. R. J. Paré. 

Evaluation of rapid solid sample extraction using the microwave-assisted process (MAPTM) 

under closed-vessel conditions. Spectros. Int. J. 13 (1), 1-14, 1997. 

19. L.D. Betowski, H.M. Webb, and A.D. Sauter. Pulsed Positive Ion Negative Ion Chemical 

Ionization Mass Spectrometric Applications to Environmental and Hazardous Waste 

Analysis. Biomed. Mass Spectrom. 10, 369, 1983. 

20. Science Applications International Corporation, "Interlaboratory Study Plan for Validation 

of Method 8276," Submitted to U.S. Environmental Protection Agency, November 2008. 

21. USEPA Region 4 Modified Method 3545A Standard Operating Procedure (SOP) for 

Pressurized Fluid Extraction (PFE), Revision 4.1, November 5, 2010. 

22. USEPA Region 4 Modified Method 3620C, SOP for Florisil® Cleanup, Revision 3.0, 

August 23, 2010. 

23. USEPA Region 4 Modified 3630C SOP for Silica Gel Cleanup, Revision 3.1, August 10, 

2011. 

24. TechLaw Incorporated, “Inter-laboratory Validation for Fish Tissue Study Plan,” 

Submitted to U.S. Environmental Protection Agency, September 2011. 

25. TechLaw Incorporated, “Inter-laboratory Validation for Fish Tissue Analytical Data 

Report,” Submitted to U.S. Environmental Protection Agency, September 2012. 
Method 8276 8276 - 29 Revision 1 

 July 2014 

26. TechLaw Incorporated, “Inter-laboratory Validation for Fish Tissue Data Validation 

Report,” Submitted to U.S. Environmental Protection Agency, September 2012. 

27. TechLaw Incorporated, “Inter-laboratory Validation for Fish Tissue Statistical Analysis 

Report,” Submitted to U.S. Environmental Protection Agency, September 2012. 

28. Parlar H, Angerhöfer D, Coelhan M, Kimmel L, "HRGC and HRGC-ECNI Determination of 

Toxaphene Residues in Fish with a New 22 Components Standard," Organohalogen 

Compd., 26, 357, 1995 

17.0 TABLES, VALIDATION DATA, FIGURES 

The following pages contain the tables and figures referenced by this method. 
Method 8276 8276 - 30 Revision 1 

July 2014 

Table 1

RECOMMENDED GC-NICI/MS OPERATING CONDITIONS FOR TOXAPHENE AND 

TOXAPHENE CONGENERS USING A NARROW-BORE CAPILLARY COLUMN1

Column - 30-m x 0.25-mm ID fused silica capillary column J&W DB-XLB, 0.25- μm film thickness. 

Carrier gas Helium 

Carrier gas pressure 10.4 psi 

Carrier gas flow 1.2 mL/min 

EPC Constant flow 

Injector temperature 205°C 

Purge flow 60 mL/min 

Purge time 1 min 

Injector mode Splitless 

Injector liner 4-mm I.D. splitless 

Transfer line temperature 280°C 

Initial oven temperature 60°C, hold 1 min 

Temperature program 60 °C to 150 °C at 10 °C/min, followed by 

150 °C to 260 °C at 3 °C/min, followed by

260 °C to 320 °C at 20 °C/min, hold 0.33 min 

followed by 320 °C to 330 °C at 50 °C/min, 

hold 3 min

Final oven temperature 330 °C 

MS mode Negative CI 

CI gas Methane 

CI gas flow 40% 

Source pressure N/A 

MS quad temperature 150 °C 

MS Source temperature 160 °C 

Solvent delay 15 min 

Acquisition mode SIM 

SIM parameters 

Resolution Low 

Dwell time 35 

Group 1 Ions 

Time, initial 306.9, 308.9, 310.9, 340.9, 342.9, 344.9, 

376.9, 378.9, 380.9, 410.9, 412.9, 414.9, 

444.8, 446.8, 448.8 

429.8 (IS), 410.8 (IS O2 rxn) 

254.9 (Surr), 497.7 (Surr) 

Scan parameters (optional) 300-500, 0.5 sec/scan 

1

Data provided courtesy of Scott Sivertsen from U S EPA Region 4 Laboratory. 
Method 8276 8276 - 31 Revision 1 

July 2014 

Table 2

SELECTED ION MONITORING FOR ANALYTES BY GC-NICI/MS 

Compound Quantitation 

Ion 

Other Ions Comments 

Toxaphene T IC1306.9, 308.9, 310.9, 340.9, 342.9, 

344.9, 376.9, 378.9, 380.9, 410.8, 

412.8, 414.8, 444.8, 446.8, 448.8 

Manual Integration 

Parlar 26 378.9 376.9, 380.9 

Parlar 40, 41 378.9 376.9, 380.9 

Parlar 50, 62 412.8 410.8, 414.8 

H x-Sed 308.9 306.9, 310.9 

H p-Sed 342.9 340.9, 344.9 

IS, PC B 204 429.8 410.8 Monitor oxygen reaction 

Surr, PC B 209 497.7 

Surr, ε-HCH 254.9 

1

Total Ion Current 
Method 8276 8276 - 32 Revision 1 

July 2014 

Table 3

SELECTED ION MONITORING FOR CHLORDANE BY GC-NICI/MS

Compound Quantitation 

Ion 

Other Ions Comments

Chlordane1 T IC2303.9, 305.9, 337.9, 339.9, 341.9, 

371.8, 373.8, 375.8, 407.8, 409.8, 

411.8, 441.8, 443.8, 445.8 

Manual integration 

IS, PC B#204 429.8 Q C check for response 

 1Ions are responses observed for technical chlordane; they may be used to confirm the presence of 

chlordane in the sample. 13 C -isotope contributions from the higher members of the chlordane congeners

contribute to monitored ions for technical toxaphene and some toxaphene congener ions. 

 2Total Ion Current 
Method 8276 8276 - 33 Revision 1 

July 2014 

2

Table 4

SELECTED ION MONITORING FOR ANALYTES BY GC-NICI/MS 

Compound Quantitation 

Ion1

Other Ions Comments 

BH C 254.9 252.9, 256.9 M -C l 

Heptachlor2 299.9 297.9, 301.9 M -C l 

Aldrin2

Dieldrin2, endrin2, endrin 

aldehyde2

Heptachlor epoxide2

329.9 327.9, 331.9 M -C l+H 

379.8 377.9, 381.9 M 

387.8 385.9, 389.9 M 

Endosulfan I, II 403.7 405.7, 407.7 M 

Endosulfan sulfate 421.7 419.7, 423.7 M 

IS, PC B 204 429.8 Q C 

1

Ions are responses observed for organochlorine pesticides; they may be used to confirm the presence of pesticides 

in the sample. These com pounds do not pose major interferences to toxaphene determination under NICI/M S 

conditions. 

2

These compounds may exhibit fragmentation resulting in a response at m /z 234.9, 236.9, and 238.9 that aids in 

confirmation. 
Method 8276 8276 - 34 Revision 1 

July 2014 

Table 5

EXAMPLE GAS CHROMATOGRAPHIC RETENTION TIMES FOR TOXAPHENE 

CONGENERS USING A NARROW-BORE CAPILLARY COLUMN (DB-XLB MSD)

Compound Retention Time (min) 

Hx-Sed 31.31 

Hp-Sed 31.95 

Parlar 26 32.38 

Parlar 41 36.69 

Parlar 40 37.03 

Parlar 44 37.35 

Parlar 50 37.89 

Parlar 62 41.91 

Decachlorobiphenyl (DCB) 48.30 
Method 8276 8276 - 35 Revision 1 

July 2014 

Table 6

QUANTITATIONS OF SPIKED SOIL FOR TOXAPHENE (25 ppb, 5-g sample) AND CHLORDANE 

(50 ppb, 5-g sample)1

Replicate No. Toxaphene (ppb) Chlordane (ppb) 

1 34.2 31.4 

2 46.4 35.2 

3 42.7 29.4 

4 35.0 41.6 

5 27.0 27.0 

6 42.0 27.8 

7 43.9 28.0 

Avg 38.7 31.5 

% RSD 17.8 16.7 

1

Data provided courtesy of Dr. Bill Brumley from US EPA ORD, 2008. Results were obtained using a 40-m Agilent J&W 0.18-

mm ID, 0.18-μm film thickness column.
Method 8276 8276 - 36 Revision 1 

July 2014 

Table 7

QUANTITATIONS OF SPIKED SOIL FOR TOXAPHENE CONGENERS (500 ppt, 5-g sample)1

Replicate No. Hx-Sed Hp-Sed Parlar 26 

1 468 532 552 

2 690 722 788 

3 768 746 778 

4 688 724 808 

5 780 752 804 

6 778 786 822 

7 672 784 706 

Avg 692 720 752 

% RSD 15.8 12.1 12.7 

Replicate No. Parlar 38, 40, 41 Parlar 44 Parlar 50, 62 

1 508 696 544 

2 792 930 812 

3 808 876 702 

4 856 1,016 844 

5 860 986 790 

6 826 942 750 

7 866 1,270 772 

Avg 788 958 744 

% RSD 16.1 17.8 13.4 

1

Data provided courtesy of Dr. Bill Brumley from US EPA ORD, 2008. Results were obtained using a 40-m Agilent J&W 0.18-

mm ID, 0.18-μm film thickness column.
Method 8276 8276 - 37 Revision 1 

July 2014 

Table 8

QUANTITATIONS OF SPIKED SOIL FOR 

ORGANOCHLORINE PESTICIDES (50 ppb, 5-g sample)1

Replicate 

No.

α-BHC β-BHC δ-BHC Heptachlor Aldrin Heptachlor 

Epoxide 

1 28.0 52.0 39.8 34.8 40.8 51.2 

2 40.6 57.6 50.0 44.6 53.4 53.0 

3 33.0 46.4 37.8 35.4 42.0 46.2 

4 27.0 71.6 40.4 28.2 34.4 51.8 

5 42.4 71.4 53.4 49.2 56.6 59.6 

6 43.2 64.4 51.2 53.0 59.8 62.4 

7 37.6 43.2 32.2 49.0 53.0 54.2 

Avg 36.0 58.0 43.6 42.0 48.6 54.0 

% RSD 18.7 19.9 18.3 22.0 19.5 10.0 

Replicate 

No. 

Endosulfan I Dieldrin Endrin Endosulfan II Endrin 

Aldehyde 

Endosulfan 

Sulfate 

1 16.8 73.8 73.6 17.7 26.0 36.6 

2 21.4 70.4 72.6 29.4 44.6 47.4 

3 37.8 61.2 63.6 38.4 19.7 37.6 

4 51.0 69.6 68.6 43.0 25.6 42.8 

5 25.2 74.4 72.8 37.8 44.2 51.0 

6 20.4 74.2 70.6 29.8 42.8 52.4 

7 13.0 62.8 66.6 11.1 37.8 46.2 

Avg 26.6 69.4 69.8 29.6 34.4 44.8 

% RSD 50.1 7.9 5.3 39.2 30.1 13.8 

1

Data provided courtesy of Dr. Bill Brumley from U S EPA O R D, 2008. Results were obtained using a 40-m Agilent J&W 

0.18-mm ID, 0.18-μm film thickness column and are included for informational purposes only to indicate that quantitation and 

acceptable recoveries of other organochlorine pesticide com pounds using GC-NICI/MS are possible. 
Method 8276 8276 - 38 Revision 1 

July 2014 

Table 9

PHASE I MULTI-LABORATORY VALIDATION DATA FOR UNKNOWN SPIKED STANDARDS1

Toxaphene 

ID No. of Labs True Value Grand Mean Std. Reproducibility

(pg/μL) (pg/μL) Deviation (RSD)

Unknown 1 9 10.0 13.0 5.7 44% 

Unknown 2 10 125 130 17.4 13% 

QC 9 150 148 10.3 7% 

H x-SED 

ID No. of Labs True Value Grand Mean Std. Reproducibility

(pg/μL) (pg/μL) Deviation (RSD)

P-Unknown 1 10 10.0 9.3 2.1 23% 

P-Unknown 2 10 125 126 10.9 9% 

H p-SED 

ID No. of Labs True Value Grand Mean Std. Reproducibility

(pg/μL) (pg/μL) Deviation (RSD)

P-Unknown 1 10 10.0 9.1 2.1 23% 

P-Unknown 2 10 125 124 9.3 7% 

P26 

ID No. of Labs True Value Grand Mean Std. Reproducibility

(pg/μL) (pg/μL) Deviation (RSD)

P-Unknown 1 10 10.0 9.7 2.7 28% 

P-Unknown 2 10 125 123 7.8 6% 

P-QC 9 200 200 22.5 11% 

P40 

ID No. of Labs True Value Grand Mean Std. Reproducibility

(pg/μL) (pg/μL) Deviation (RSD)

P-Unknown 1 9 10.0 9.4 2.6 28% 

P-Unknown 2 9 125 121 9.0 7% 

P-QC 8 200 182 22.5 12% 
Method 8276 8276 - 39 Revision 1 

July 2014 

Table 9 (cont.)

PHASE I MULTI-LABORATORY VALIDATION DATA FOR UNKNOWN SPIKED STANDARDS

P41 

ID No. of Labs True Value Grand Mean Std. Reproducibility

(pg/μL) (pg/μL) Deviation (RSD)

P-Unknown 1 9 10.0 9.6 3.8 40% 

P-Unknown 2 9 125 121 9.8 8% 

P-QC 8 200 176 22.0 13% 

P44 

ID No. of Labs True Value Grand Mean Std. Reproducibility

(pg/μL) (pg/μL) Deviation (RSD)

P-Unknown 1 10 10.0 9.2 3.0 33% 

P-Unknown 2 10 125 121 15.0 12% 

P-QC 9 200 269 41.9 16% 

P50 

ID No. of Labs True Value Grand Mean Std. Reproducibility

(pg/μL) (pg/μL) Deviation (RSD)

P-Unknown 1 10 10.0 9.1 2.9 32% 

P-Unknown 2 10 125 122 9.2 8% 

P-QC 9 200 182 22.0 12% 

P62 

ID No. of Labs True Value Grand Mean Std. Reproducibility

(pg/μL) (pg/μL) Deviation (RSD)

P-Unknown 1 10 10.0 7.1 2.7 38% 

P-Unknown 2 10 125 127 19.4 15% 

P-QC 9 200 320 62.0 19% 

1

The values of the quality control standards were unknown to the analysts.
Method 8276 8276 - 40 Revision 1 

July 2014 

Table 10

PHASE II MULTI-LABORATORY VALIDATION DATA USING SPIKED EXTRACTS OF 

UNCONTAMINATED REAL WORLD SAMPLE MATRICES1

Toxaphene

ID No. of Labs True Value Grand Mean Std. Reproducibility

(pg/μL) (pg/μL) Deviation (RSD)

GW 2 9 600 694 58.6 8.4% 

Sed 2 9 700 881 159 18.0% 

Soil 2 9 400 458 71.0 15.5% 

Hx-SED

ID No. of Labs True Value

(pg/μL)

Grand Mean

(pg/μL)

Std. 

Deviation

Reproducibility

(RSD) 

GW 1 9 1.0 0.98 0.11 11.2% 

GW 2 9 150 146 9.2 6.3% 

Sed 1 9 4.0 3.57 0.36 10.1% 

Sed 2 9 200 198 22.0 11.1% 

Soil 1 9 2.0 1.92 0.26 13.5% 

Soil 2 9 250 253 25.7 10.2% 

Hp-SED

ID No. of Labs True Value

(pg/μL)

Grand Mean

(pg/μL)

Std. 

Deviation

Reproducibility

(RSD) 

GW 1 9 1.0 1.00 0.11 11.0% 

GW 2 9 150 148 7.9 5.3% 

Sed 1 9 4.0 3.61 0.44 12.2% 

Sed 2 9 200 198 18.9 9.5% 

Soil 1 9 2.0 1.99 0.26 13.1% 

Soil 2 9 250 255 24.1 9.5% 

1

Liquid samples were solvent extracted with methylene chloride according to Method 3510 using a sample volume of one 

L and concentrated to a final extract volume of 5 mL in isooctane. Solid samples were solvent extracted using 

methylene chloride according to Method 3540 with a sample mass of 30-g and concentrated to a final extract volume of 

5 mL in isooctane. 
Method 8276 8276 - 41 Revision 1 

July 2014 

Table 10 (cont.)

PHASE II MULTI-LABORATORY VALIDATION DATA USING SPIKED EXTRACTS OF 

UNCONTAMINATED REAL WORLD SAMPLE MATRICES1

P26 

ID No. of Labs True Value

(pg/μL)

Grand Mean

(pg/μL)

Std. 

Deviation

Reproducibility

(RSD) 

GW 1 9 1.0 0.96 0.12 12.5% 

GW 2 9 150 145 6.8 4.7% 

Sed 1 9 4.0 3.56 0.34 9.6% 

Sed 2 9 200 196 17.9 9.1% 

Soil 1 9 2.0 1.95 0.27 13.8% 

Soil 2 9 250 257 23.0 8.9% 

P40 

ID No. of Labs True Value

(pg/μL)

Grand Mean

(pg/μL)

Std. 

Deviation

Reproducibility

(RSD) 

GW 1 9 1.0 1.03 0.15 14.6% 

GW 2 9 150 149 7.1 4.8% 

Sed 1 9 4.0 3.72 0.47 12.6% 

Sed 2 9 200 203 20.7 10.2% 

Soil 1 9 2.0 2.11 0.25 11.8% 

Soil 2 9 250 267 26.6 10.0% 

P41 

ID No. of Labs True Value

(pg/μL)

Grand Mean

(pg/μL)

Std. 

Deviation

Reproducibility

(RSD) 

GW 1 9 1.0 0.97 0.12 12.4% 

GW 2 9 150 146 9.0 6.2% 

Sed 1 9 4.0 3.68 0.46 12.5% 

Sed 2 9 200 196 23.8 12.1% 

Soil 1 9 2.0 2.04 0.28 13.7% 

Soil 2 9 250 263 27.3 10.4% 

1 Liquid samples were solvent extracted with methylene chloride according to Method 3510 using a sample volume of one 

L and concentrated to a final extract volume of 5 mL in isooctane. Solid samples were solvent extracted using methylene 

chloride according to Method 3540 with a sample mass of 30-g and concentrated to a final extract volume of 5 mL in 

isooctane. 
Method 8276 8276 - 42 Revision 1 

July 2014 

Table 10 (cont.)

PHASE II MULTI-LABORATORY VALIDATION DATA USING SPIKED EXTRACTS OF 

UNCONTAMINATED REAL WORLD SAMPLE MATRICES1

P44 

ID No. of Labs True Value

(pg/μL)

Grand Mean

(pg/μL)

Std. 

Deviation

Reproducibility

(RSD) 

GW 1 9 1.0 0.93 0.18 19.4% 

GW 2 9 150 149 13.1 8.8% 

Sed 1 9 4.0 3.91 1.05 26.9% 

Sed 2 9 200 208 34.4 16.5% 

Soil 1 9 2.0 2.01 0.30 14.9% 

Soil 2 9 250 282 32.1 11.4% 

P50 

ID No. of Labs True Value

(pg/μL)

Grand Mean

(pg/μL)

Std. 

Deviation

Reproducibility

(RSD) 

GW 1 9 1.0 0.97 0.18 18.6% 

GW 2 9 150 148 8.0 5.4% 

Sed 1 9 4.0 3.68 0.44 12.0% 

Sed 2 9 200 199 21.2 10.7% 

Soil 1 9 2.0 2.00 0.27 13.5% 

Soil 2 9 250 265 22.7 8.6% 

P62 

ID No. of Labs True Value

(pg/μL)

Grand Mean

(pg/μL)

Std. 

Deviation

Reproducibility

(RSD) 

GW 1 9 1.0 0.96 0.33 34.4% 

GW 2 9 150 143 18.6 13.0% 

Sed 1 9 4.0 4.09 1.08 26.4% 

Sed 2 9 200 198 44.5 22.5% 

Soil 1 9 2.0 2.02 0.29 14.4% 

Soil 2 9 250 273 57.9 21.2% 

1 Liquid samples were solvent extracted with methylene chloride according to Method 3510 using a sample volume of one 

L and concentrated to a final extract volume of 5 mL in isooctane. Solid samples were solvent extracted using methylene 

chloride according to Method 3540 with a sample mass of 30-g and concentrated to a final extract volume of 5 mL in 

isooctane. 
Method 8276 8276 - 43 Revision 1 

July 2014 

Table 11

PHASE II MULTI-LABORATORY VALIDATION DATA USING REAL WORLD SAMPLES FROM 

TERRY CREEK IN BRUNSWICK, GEORGIA1

SEDIMENT 

ID N o. of Labs G rand Mean

(pg/μL) 

G rand Mean

(mg/kg) 

Std. Deviation

(pg/μL) 

Reproducibility

(R SD ) 

Toxaphene 9 173,000 36,300 91,000 53% 

Hx-Sed 9 3,600 754 366 10% 

Hp-Sed 9 3,700 776 526 14% 

P26 8 1,250 261 163 13% 

P40 9 2,660 557 951 36% 

P41 9 1,310 273 1530 117% 

P44 8 902 189 172 19% 

P50 9 1,780 372 231 13% 

P62 8 3,840 803 1,410 37% 

SO IL

ID N o. of Labs G rand Mean

(pg/μL) 

G rand Mean

(mg/kg) 

Std. Deviation

(pg/μL) 

Reproducibility

(R SD ) 

Toxaphene 9 2,980 624 1,730 58% 

Hx-Sed 9 153 32 37 24% 

Hp-Sed 9 129 27 30 23% 

P26 8 6.74 1.4 2.08 31% 

P40 9 15.0 3.1 7.4 49% 

P41 9 6.0 1.3 8.5 142% 

P44 8 2.53 0.5 1.30 51% 

P50 9 4.0 0.8 1.2 30% 

P62 8 20.7 4.3 45.8 221% 

1

Samples were solvent extracted using methylene chloride according to Method 3540 with a sample mass of 30 g 

and concentrated to a final extract volume of 5 mL in isooctane. 
Method 8276 8276 - 44 Revision 1 

July 2014 

Table 12

PHASE II MULTI-LABORATORY VALIDATION DATA USING REAL WORLD SAMPLES FROM 

TERRY CREEK IN BRUNSWICK, GEORGIA WITH EXTRACTS SUBJECTED TO CLEANUP1

Technical Toxaphene

ID No. of Labs True Value Grand Mean Grand Mean % Rec. Std. Dev. Reproducibility

(pg/μL) (pg/μL) (mg/kg) (pg/μL) (RSD) 

TC Sed 4 --- 305,000 63,900 --- 47,200 15%

TC Soil 4 --- 15,600 3,260 --- 4,570 29% 

PCB 209 Sed Surr 4 100 106 --- 106% 4.0 4% 

ε -HCH Sed Surr 4 250 231 --- 92% 21.0 9% 

PCB 209 Soil Surr 4 100 106 --- 106% 1.0 1%

ε -HCH Soil Surr 4 250 240 --- 96% 11.8 5%

LCS 4 200 228 --- 114% 27.5 12

Hx-SED

ID No. of Labs True Value Grand Mean Grand Mean % Rec. Std. Dev. Reproducibility

TC Sed 4 --- 3,680 772 --- 183 5%

TC Soil 4 --- 1,380 290 --- 91 7% 

PCB 209 Sed Surr 4 100 106 --- 106% 4.5 4% 

ε -HCH Sed Surr 4 250 234 --- 94% 11.9 5% 

PCB 209 Soil Surr 4 100 121 --- 121% 9.7 8% 

ε -HCH Soil Surr 4 250 246 --- 98% 4.8 2% 

LCS 4 200 211 --- 105% 12.8 6% 

Hx-SED

ID No. of Labs True Value Grand Mean Grand Mean % Rec. Std. Dev. Reproducibility

TC Sed 4 --- 4,220 883 --- 993 24%

TC Soil 4 --- 1,100 231 --- 29 3% 

PCB 209 Sed Surr 4 100 106 --- 106% 4.5 4% 

ε -HCH Sed Surr 4 250 234 --- 94% 11.9 5% 

PCB 209 Soil Surr 4 100 121 --- 121% 9.7 8% 

ε -HCH Soil Surr 4 250 246 --- 98% 4.8 2% 

LCS 4 200 212 --- 106% 14.2 7% 

 1Samples were solvent extracted using methylene chloride according to Method 3540 with a sample mass of 30 g 

and concentrated to a volume of 10 mL. These extracts were further subjected to clean-up using a copper, acid, and 

silica gel approach according to Methods 3660, 3665, and 3630, respectively, to a final extract volume of 5 mL in 

isooctane. 
Method 8276 8276 - 45 Revision 1 

July 2014 

Table 12 (cont.)

PHASE II MULTI-LABORATORY VALIDATION DATA USING REAL WORLD SAMPLES FROM 

TERRY CREEK IN BRUNSWICK, GEORGIA WITH EXTRACTS SUBJECTED TO CLEANUP1

P26 

ID No. of Labs True Value Grand Mean Grand Mean % Rec. Std. Dev. Reproducibility

(pg/μL) (pg/μL) (mg/kg) (pg/μL) (RSD) 

TC Sed 4 --- 1,870 392 --- 156 8%

TC Soil 4 --- 60 13 --- 6 11% 

PCB 209 Sed Surr 4 100 106 --- 106% 4.5 4% 

ε -HCH Sed Surr 4 250 234 --- 94% 11.9 5% 

PCB 209 Soil Surr 4 100 121 --- 121% 9.7 8% 

ε -HCH Soil Surr 4 250 246 --- 98% 4.8 2% 

LCS 4 200 216 --- 108% 22.8 11% 

P40 

ID No. of Labs True Value Grand Mean Grand Mean % Rec. Std. Dev. Reproducibility

(pg/μL) (pg/μL) (mg/kg) (pg/μL) (RSD) 

TC Sed 4 --- 4,200 880 --- 1,890 45%

TC Soil 4 --- 143 30 --- 60 42% 

PCB 209 Sed Surr 4 100 106 --- 106% 4.5 4% 

ε -HCH Sed Surr 4 250 234 --- 94% 11.9 5% 

PCB 209 Soil Surr 4 100 121 --- 121% 9.7 8% 

ε -HCH Soil Surr 4 250 246 --- 98% 4.8 2% 

LCS 4 200 219 --- 110% 24.3 11% 

P41 

ID No. of Labs True Value Grand Mean Grand Mean % Rec. Std. Dev. Reproducibility

(pg/μL) (pg/μL) (mg/kg) (pg/μL) (RSD) 

TC Sed 4 --- 1,230 258 --- 175 14%

TC Soil 4 --- 33 7 --- 17 51% 

PCB 209 Sed Surr 4 100 106 --- 106% 4.5 4% 

ε -HCH Sed Surr 4 250 234 --- 94% 11.9 5% 

PCB 209 Soil Surr 4 100 121 --- 121% 9.7 8% 

ε -HCH Soil Surr 4 250 246 --- 98% 4.8 2% 

LCS 4 200 220 --- 110% 28.1 13% 

 1Samples were solvent extracted using methylene chloride according to Method 3540 with a sample mass of 30 g 

and concentrated to a volume of 10 mL. These extracts were further subjected to clean-up using a copper, acid, and 

silica gel approach according to Methods 3660, 3665, and 3630, respectively, to a final extract volume of 5 mL in 

isooctane. 
Method 8276 8276 - 46 Revision 1 

July 2014 

Table 12 (cont.)

PHASE II MULTI-LABORATORY VALIDATION DATA USING REAL WORLD SAMPLES FROM 

TERRY CREEK IN BRUNSWICK, GEORGIA WITH EXTRACTS SUBJECTED TO CLEANUP1

P44 

ID No. of Labs True Value Grand Mean Grand Mean % Rec. Std. Dev. Reproducibility

(pg/μL) (pg/μL) (mg/kg) (pg/μL) (RSD) 

TC Sed 3 --- 1,390 292 --- 194 14%

TC Soil 3 --- 30 6 --- 5 18% 

PCB 209 Sed Surr 4 100 106 --- 106% 4.5 4% 

ε -HCH Sed Surr 4 250 234 --- 94% 11.9 5% 

PCB 209 Soil Surr 4 100 121 --- 121% 9.7 8% 

ε -HCH Soil Surr 4 250 246 --- 98% 4.8 2% 

LCS 4 200 209 --- 104% 29.8 14% 

P50 

ID No. of Labs True Value Grand Mean Grand Mean % Rec. Std. Dev. Reproducibility

(pg/μL) (pg/μL) (mg/kg) (pg/μL) (RSD) 

TC Sed 4 --- 2,920 612 --- 342 12%

TC Soil 4 --- 69 14 --- 7 11% 

PCB 209 Sed Surr 4 100 106 --- 106% 4.5 4% 

ε -HCH Sed Surr 4 250 234 --- 94% 11.9 5% 

PCB 209 Soil Surr 4 100 121 --- 121% 9.7 8% 

ε -HCH Soil Surr 4 250 246 --- 98% 4.8 2% 

LCS 4 200 221 --- 110% 23.8 11% 

P62 

ID No. of Labs True Value Grand Mean Grand Mean % Rec. Std. Dev. Reproducibility

(pg/μL) (pg/μL) (mg/kg) (pg/μL) (RSD) 

TC Sed 3 --- 8,840 1,850 --- 1,550 18%

TC Soil 3 --- 219 46 --- 54 24% 

PCB 209 Sed Surr 4 100 106 --- 106% 4.5 4% 

ε -HCH Sed Surr 4 250 234 --- 94% 11.9 5% 

PCB 209 Soil Surr 4 100 121 --- 121% 9.7 8% 

ε -HCH Soil Surr 4 250 246 --- 98% 4.8 2% 

LCS 4 200 226 --- 113% 27.6 12% 

1 Samples were solvent extracted using methylene chloride according to Method 3540 with a sample mass of 30 g and 

concentrated to a volume of 10 mL. These extracts were further subjected to clean-up using a copper, acid, and 

silica gel approach according to Methods 3660, 3665, and 3630, respectively, to a final extract volume of 5 mL in 

isooctane. 
Method 8276 8276 - 47 Revision 1 

July 2014 

Table 13

PHASE II MULTI-LABORATORY VALIDATION DATA COMPARISON USING REAL WORLD 

SAMPLES FROM TERRY CREEK IN BRUNSWICK, GEORGIA (EXTRACTION ONLY COMPARED 

TO EXTRACTS SUBJECTED TO CLEANUP)

SEDIMENT 

ID N o. of Labs

1 N o. of Labs2 G rand Mean G rand Mean G rand Mean G rand Mean

Extraction only Cleanup 

(pg/μL)

1

Extraction only

(pg/μL)

2

Cleanup 

(mg/kg)

1

Extraction only

(mg/kg)

2

Cleanup 

T oxaphene 9 4 173,000 305,000 36,300 63,900 

H x-Sed 9 4 3,600 3,680 754 772 

H p-Sed 9 4 3,700 4,220 776 883 

P26 8 4 1,250 1,870 261 392 

P40 9 4 2,660 4,200 557 880 

P41 9 4 1,310 1,230 273 258 

P44 8 3 902 1,390 189 292 

P50 9 4 1,780 2,920 372 612 

P62 8 3 3,840 8,840 803 1,850 

SO IL

ID N o. of Labs

1 N o. of Labs2 G rand Mean

(pg/μL)

1

G rand Mean

(pg/μL)

2

G rand Mean

(mg/kg)

1

G rand Mean

(mg/kg)

2

Extraction only Cleanup Extraction only Cleanup Extraction only Cleanup

Toxaphene 9 4 2,980 15,600 624 3,260 

Hx-Sed 9 4 153 1,380 32 290 

Hp-Sed 9 4 129 1,100 27 231 

P26 8 4 6.74 60 1.4 13 

P40 9 4 15.0 143 3.1 30 

P41 9 4 6.0 33 1.3 7 

P44 8 3 2.53 30 0.5 6 

P50 9 4 4.0 69 0.8 14 

P62 8 3 20.7 219 4.3 46 

1 Samples were solvent extracted using methylene chloride according to Method 3540 with a sample mass of 30 g and 

concentrated to a final extract volume of 5 mL in isooctane. 

2 Samples were solvent extracted using methylene chloride according to Method 3540 with a sample mass of 30 g and 

concentrated to a volume of 10 mL. These extracts were further subjected to clean-up using a copper, acid, and silica 

gel approach according to Methods 3660, 3665, and 3630, respectively, to a final extract volume of 5 mL in isooctane. 
Method 8276 8276 - 48 Revision 1 

July 2014 

Table 14

PHASE II MULTI-LABORATORY VALIDATION DATA USING MULTIPLE TECHNICAL 

TOXAPHENE SPIKING PROTOCOLS FOR UNCONTAMINATED REAL WORLD SAMPLE 

MATRICES1

Technical Toxaphene

Spiked Prior to Extraction and Cleanup

ID No. of Labs True Value

(pg/μL)

Grand Mean

(pg/μL)

% Recovery Std. 

Deviation

(pg/μL)

Reproducibility

RSD 

Sed 1L 4 75 78 105% 6.9 9% 

PCB 209 Surr 4 100 109 109% 2 2% 

ε -HCH Surr 4 250 275 110% 10 4% 

Sed 1M 4 150 148 99% 16 11% 

PCB 209 Surr 4 100 97 97% 5 5% 

ε -HCH Surr 4 250 238 95% 17 7% 

LCS L 4 75 71 95% 9 12% 

LCS M 4 200 185 93% 27 15% 

Low-level Check 4 40 49 121% 7 14% 

Technical Toxaphene

Spiked After Extraction and Prior to Clean-up

ID No. of Labs True Value

(pg/μL)

Grand Mean

(pg/μL)

% Recovery Std. 

Deviation

(pg/μL)

Reproducibility

RSD 

Sed 2L 4 75 73 97% 9.1 12% 

PCB 209 Surr 4 100 100 100% 4 4% 

ε -HCH Surr 4 250 258 103% 14 5% 

Sed 2M 4 150 153 102% 18 12% 

PCB 209 Surr 4 100 99 99% 6 6% 

ε -HCH Surr 4 250 255 102% 19 7% 

LCS L 4 75 71 95% 9 12% 

LCS M 4 200 185 93% 27 15% 

Low-level Check 4 40 49 121% 7 14% 

1

Samples were solvent extracted using methylene chloride according to Method 3540 with a sample mass of 30 g 

and concentrated to a volume of 10 mL. These extracts were further subjected to clean-up using a copper, acid, and 

silica gel approach according to Methods 3660, 3665, and 3630, respectively, to a final extract volume of 5 mL in 

isooctane. 

Table 14 
Method 8276 8276 - 49 Revision 1 

July 2014 

(cont.)

PHASE II MULTI-LABORATORY VALIDATION DATA USING MULTIPLE SPIKING 

PROTOCOLS FOR UNCONTAMINATED REAL WORLD SAMPLE MATRICES1

Technical 

Toxaphene

Spiked in the Final 

Extract

ID No. of Labs True Value

(pg/μL)

Grand Mean

(pg/μL)

% Recovery Std. Deviation

(pg/μL)

Reproducibility

RSD 

Sed 3L 4 75 86 114% 10 12% 

PCB 209 Surr 4 100 105 105% 5 5% 

ε -HCH Surr 4 250 276 111% 21 7% 

Sed 3M 4 150 157 105% 21 13% 

PCB 209 Surr 4 100 105 105% 5 5% 

ε-HCH Surr 4 250 273 109% 18 7% 

LCS L 4 75 71 95% 9 12% 

LCS M 4 200 185 93% 27 15% 

Low-level Check 4 40 49 121% 7 14% 

 1Samples were solvent extracted using methylene chloride according to Method 3540 with a sample mass of 30 g 

and concentrated to a volume of 10 mL. These extracts were further subjected to clean-up using a copper, acid, 

and silica gel approach according to Methods 3660, 3665, and 3630, respectively, to a final extract volume of 5 

mL in isooctane. 

 
Method 8276 8276 - 50 Revision 1 

July 2014 

Table 15 

PHASE III MULTI-LABORATORY VALIDATION DATA USING SPIKED LABORATORY QUALITY 

CONTROL SAMPLES CARRIED THROUGH EXTRACTION AND CLEANUP1

Toxaphene 

ID No. of 

Participants 

True 

Value 

(pg/μL) 

Grand 

Mean 

(pg/μL) 

% 

Recovery 

Std. 

Deviation 

Reproducibility 

(% RSD) 

Second Source 

Standard 8 150 159 106 9.9 6.2 

LCS 1 8 50 57.3 115 9.7 16.9 

LCS 2 8 200 207 104 13.4 6.5 

PCB 209 LCS1 8 100 95.0 95.0 1.9 2.0 

ε-HCH LCS 1 8 250 231 92.2 21.0 9.1 

PCB 209 LCS2 8 100 90.2 90.2 5.4 6.0 

ε-HCH LCS2 8 250 240 95.8 15.4 6.4 

Hx-Sed 

ID No. of 

Participants 

True Value 

(pg/μL) 

Grand Mean 

(pg/μL) 

% 

Recovery 

Std. 

Deviation 

Reproducibility 

(% RSD) 

LCS1 8 100 98.4 98.4 5.84 5.94 

LCS2 8 2 1.95 97.4 0.16 8.00 

Hp-Sed 

ID No. of 

Participants 

True Value 

(pg/μL) 

Grand Mean 

(pg/μL) 

% 

Recovery 

Std. 

Deviation 

Reproducibility 

 (% RSD) 

LCS1 8 100 98.9 98.9 5.77 5.84 

LCS2 8 2 1.99 99.3 0.13 6.30 

P26 

ID No. of 

Participants 

True Value 

(pg/μL) 

Grand Mean 

(pg/μL) 

% 

Recovery 

Std. 

Deviation 

Reproducibility 

(% RSD) 

LCS1 8 100 97.9 97.9 5.27 5.39 

LCS2 8 2 1.83 91.4 0.36 19.7 
Method 8276 8276 - 51 Revision 1 

July 2014 

Table 15 (cont.) 

P40 

ID No. of 

Participants 

True Value 

(pg/μL) 

Grand Mean 

(pg/μL) 

% 

Recovery 

Std. 

Deviation 

Reproducibility 

(% RSD) 

LCS1 8 100 98.1 98.1 6.33 6.45 

LCS2 8 2 1.94 97.2 0.15 7.46 

P41 

ID No. of 

Participants 

True Value 

(pg/μL) 

Grand Mean 

(pg/μL) 

% 

Recovery 

Std. 

Deviation 

Reproducibility 

 (% RSD) 

LCS1 8 100 98.6 98.6 6.31 6.40 

LCS2 8 2 2.03 102 0.17 8.32 

P44 

ID No. of 

Participants 

True Value 

(pg/μL) 

Grand Mean 

(pg/μL) 

% 

Recovery 

Std. 

Deviation 

Reproducibility 

(RSD) 

LCS1 8 100 99.4 99.4 8.85 8.91 

LCS2 8 2 1.99 99.7 0.16 8.09 

P50 

ID No. of 

Participants 

True Value 

(pg/μL) 

Grand Mean 

(pg/μL) 

% 

Recovery 

Std. 

Deviation 

Reproducibility 

(% RSD) 

LCS1 8 100 96.9 96.9 7.47 7.71 

LCS2 8 2 2.01 101 0.07 3.55 

P62 

ID No. of 

Participants 

True Value 

(pg/μL) 

Grand Mean 

(pg/μL) 

% 

Recovery 

Std. 

Deviation 

Reproducibility 

(% RSD) 

LCS1 8 226 237.0 105 26.0 11.0 

LCS2 8 22.6 21.0 92.9 4.51 21.5 

Table 15 (cont.)
Method 8276 8276 - 52 Revision 1 

July 2014 

Congeners - PCB 209

ID No. of 

Participants 

True Value 

(pg/μL) 

Grand Mean 

(pg/μL) 

% 

Recovery 

Std. 

Deviation 

Reproducibility 

(% RSD) 

LCS1 8 100 95.1 95.1 5.13 5.39 

LCS2 8 100 94.9 94.9 5.55 5.85 

Congeners – ε-HCH

ID No. of 

Participants 

True Value 

(pg/μL) 

Grand Mean 

(pg/μL) 

% 

Recovery 

Std. 

Deviation 

Reproducibility 

(% RSD) 

LCS1 8 250 248.0 99.0 11.7 4.72 

LCS2 8 250 247.0 98.9 7.84 3.17 

1Samples were extracted in hexane in an Accelerated Solvent Extractor (ASE) using a modified version of

Method 3545A with a sample mass of 10 g of solid material (Ottawa sand). In-cell cleanup was done in the 

ASE extraction cell using activated, acidified (30% H2SO4) silica gel per Method 3665A and Method 3630C. 

These extracts were further subjected to cleanup using a Florisil® cartridge (1% deactivated with DI water) 

approach according to Method 3620C. The sample was in hexane throughout the process and was concentrated 

to a final extract volume of 1 mL. 

 
Method 8276 8276 - 53 Revision 1 

July 2014 

Table 16 

PHASE III MULTI-LABORATORY VALIDATION DATA USING FISH SAMPLES COLLECTED FROM 

TERRY CREEK IN BRUNSWICK, GEORGIA WITH EXTRACTS SUBJECTED TO CLEANUP1

Toxaphene 

ID No. of 

Participants Grand Mean (pg/μL) Std. Deviation 

Reproducibility 

% RSD 

TC 1 8 351 71.9 20.5 

TC 2 8 317 79.3 25.0 

Hp-Sed

ID No. of 

Participants 

Grand Mean 

 (pg/μL) 

Std. 

Deviation 

Reproducibility 

% RSD 

TC1 8 91.8 5.66 6.17 

TC2 8 75.6 5.80 7.67 

P26

ID No. of 

Participants 

Grand Mean 

(pg/μL) 

Std. 

Deviation 

Reproducibility 

% RSD 

TC1 8 8.05 0.40 4.93 

TC2 8 11.6 0.52 4.46 

P40

ID No. of 

Participants 

Grand Mean 

(pg/μL) 

Std. 

Deviation 

Reproducibility 

% RSD 

TC1 8 11.1 1.02 9.19 

TC2 8 12.6 0.71 5.66 

 

Hx-Sed

ID No. of Participants Grand Mean 

(pg/μL) 

Std. 

Deviation 

Reproducibility 

% RSD 

TC1 8 80.3 5.59 6.97 

TC2 8 54.2 4.49 8.27 
Method 8276 8276 - 54 Revision 1 

July 2014 

Table 16 (cont.) 

P41

ID No. of 

Participants 

Grand Mean 

(pg/μL) 

Std. 

Deviation 

Reproducibility 

% RSD 

TC1 8 4.49 0.19 4.31 

TC2 8 5.29 0.21 3.90 

P44

ID No. of 

Participants 

Grand Mean 

(pg/μL) 

Std. 

Deviation 

Reproducibility 

% RSD 

TC1 8 4.76 0.33 6.86 

TC2 8 6.60 0.55 8.33 

P50

ID No. of 

Participants 

Grand Mean 

(pg/μL) 

Std. 

Deviation 

Reproducibility 

% RSD 

TC1 8 8.34 0.73 8.76 

TC2 8 12.9 1.06 8.18 

P62

ID No. of 

Participants 

Grand Mean 

(pg/μL) 

Std. 

Deviation 

Reproducibility 

% RSD 

TC1 8 10.5 2.81 26.9 

TC2 8 14.8 4.13 27.8 

1Homogenized fish tissue samples were extracted in hexane in an Accelerated Solvent 

Extractor (ASE) using a modified version of Method 3545. In-cell cleanup was done in the 

ASE extraction cell using activated, acidified (30% H2SO4) silica gel per Method 3665 and 

Method 3630. These extracts were further subjected to cleanup using a Florisil® cartridge 

(1% deactivated with deionized water) approach according to Method 3620. The sample was 

in hexane throughout the process. Because of varying concentrations of incurred toxaphene 

residues in the tissue samples, extraction sample masses were varied and final extract 

volumes were adjusted based on sample knowledge and instrument calibration ranges.

 
Method 8276 8276 - 55 Revision 1 

July 2014 

FIGURE 1 

EXAMPLE GAS CHROMATOGRAM OF A MIXED CONGENER STANDARD

 
Method 8276 8276 - 56 Revision 1 

July 2014 

FIGURE 2

EXAMPLE GAS CHROMATOGRAM OF A FISH TISSUE EXTRACT 

 
Method 8276 8276 - 57 Revision 1 

July 2014 

FIGURE 3 

EXAMPLE GAS CHROMATOGRAM OF A 100 pg/μL CONGENER CALIBRATION VERIFICATION 

STANDARD 

 
Method 8276 8276 - 58 Revision 1 

July 2014 

FIGURE 4 

EXAMPLE GAS CHROMATOGRAM OF AN EPA REGION 4 SEDIMENT EXTRACT SPIKED USING 

DECACHLOROBIPHENYL 

 
Method 8276 8276 - 59 Revision 1 

July 2014 

Appendix A: Changes in Revision 1 from the Revision 0, 2010 Version. 

 

1. The acronym NIMS (negative ion mass spectroscopy) has been replaced with NICI/MS (negative 

ion chemical ionization/mass spectrometry). 

2. Section 2.0 now points to Method 3500 for sample preparation guidance, rather than listing all 

methods specifically. 

3. References to the method quantitation limit (MQL) have been replaced with the lower limit of 

quantitation (LLOQ). 

4. Section 6.4.1 no longer specifies exact mass spectrometer conditions. Instead, it states that the 

detector must tuned per Section 11.4.1 and Table 1 conditions. 

5. Sections 9.4 (IDP) and 9.7 (LLOQ) were expanded to reflect current guidance. 

6. Section 11.1 was re-organized into tabular form to increase clarity and ease of preparatory method 

selection. 

7. Improved overall method formatting for consistency with new SW-846 methods style guidance. 

The format was updated to Microsoft Word .docx. Many minor editorial and technical revisions were 

made throughout to improve method clarity. 

8. The revision number was changed to one and the date published was changed to July 2014. 

9. This appendix was added showing changes from the previous revision.