An American National Standard

Designation: D 5810 – 96 (Reapproved 2001)

Standard Guide for

Spiking into Aqueous Samples 1
This standard is issued under the fixed designation D 5810; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (e) indicates an editorial change since the last revision or reapproval.

1. Scope recovery data cannot be obtained if an aqueous solution or

1.1 This guide covers the general technique of “spiking” a homogenous suspension of the analyte of interest in the sample
broad range of materials into aqueous media. This guide will cannot be attained. These procedures may be applicable to
serve the analyst in preparing spiked samples for quality microbiological preparations if the homogeneity of the suspen-
control purposes. Guidance is also provided to aid the analyst sion can be adequately maintained throughout the course of the
in calculating recoveries and interpreting results. It is the analysis, for example, by mechanical agitation or stirring.
responsibility of the analyst to determine whether the proce- 1.5 Matrix spiking may be performed in the field or in the
dures and materials described here are appropriate to the task laboratory, depending on which part of the analytical process is
at hand. to be tested. Field spiking tests the recovery of the overall
1.2 The procedures in this guide are focused on “matrix process, including preservation and shipping of the sample.
spike” preparation, analysis, and interpretation of results. The Laboratory spiking tests the laboratory process only. Spiking of
applicability of these procedures to the preparation of calibra- sample extracts, concentrates, or dilutions will test only that
tion standards, calibration check standards, laboratory control portion of the process subsequent to addition of the spike.
standards, reference materials, and other quality control mate- 1.6 The values stated in SI units are to be regarded as the
rials by spiking is incidental. A sample (the matrix) is fortified standard.
(spiked) with the analyte of interest for a variety of analytical 1.7 This standard does not purport to address all of the
and quality control purposes. While the spiking of multiple safety concerns, if any, associated with its use. It is the
sample portions is discussed, the method of standard additions responsibility of the user of this standard to establish appro-
is not covered. priate safety and health practices and determine the applica-
1.3 This guide is intended for use in conjunction with the bility of regulatory limitations prior to use.
individual analytical test method that provides procedures for 2. Referenced Documents
analysis of the analyte or component of interest. The test
method is used to determine an analyte or component’s 2.1 ASTM Standards:
background level and, again after spiking, its now elevated D 1129 Terminology Relating to Water2
level. Each test method typically provides procedures not only D 1193 Specification for Reagent Water2
for samples, but also for calibration standards or analytical D 3694 Practices for Preparation of Sample Containers and
control solutions, or both. These procedures include prepara- for Preservation of Organic Constituents3
tion, handling, storage, preservation, and analysis techniques. D 3856 Guide for Good Laboratory Practices in Laborato-
These procedures are applicable by extension, using the ries Engaged in Sampling and Analysis of Water2
analyst’s judgement on a case-by-case basis, to spiking solu- D 4375 Practice for Basic Statistics in Committee D19 on
tions, and are not reiterated in this guide. See also Practice Water2
E 200 for preparation and storage information. E 200 Practice for Preparation, Standardization, and Stor-
1.4 These procedures apply only to analytes that are soluble age of Standard and Reagent Solutions for Chemical
in water at the concentration of the spike plus any background Analysis2
material, or to analytes soluble in a solvent that is itself 3. Terminology
water-soluble. The system used in the later case must result in
a homogeneous solution of analyte and sample. Meaningful 3.1 Definitions—For definitions of terms used in this guide,
refer to Terminology D 1129.

1
This guide is under the jurisdiction of ASTM Committee D19 on Water and is
the direct responsibility of Subcommittee D19.02 on General Specifications,
2
Technical Resources, and Statistical Methods. Annual Book of ASTM Standards, Vol 11.01.
3
Current edition approved Jan. 10, 1996. Published March 1996. Annual Book of ASTM Standards, Vol 11.02.

Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.

1
 D 5810 – 96 (2001)
3.2 Definitions of Terms Specific to This Standard: Training of field personnel and validation of their spiking
3.2.1 matrix spike, n—the quantity (mass) of a component techniques are necessary to ensure that spikes are added
(analyte) of interest that is added to a sample (matrix) in order accurately and reproducibly. Duplicate field spikes can be used
to test the bias as measured by recovery (of that component to document the reproducibility of the technique. When envi-
under specific analytical conditions) and reported as percent ronmentally labile compounds are used as spikes, the spiking
recovery (P). solution shall be protected up to the point of use by appropriate
3.2.2 spike, v—the addition of a known amount of an means such as chilling, protection from sunlight and oxygen, or
analyte of known identity to a measured volume of a sample chemical preservation.
(from a specific matrix) to determine the efficiency with which NOTE 1—Any field spiked sample, if known to the laboratory, should be
the added analyte can be “recovered” from (measured in) that labeled as a field spike in the final results report. Also, whenever possible,
matrix by the analytical system after exposure to a specific field spiking of volatile compounds should be avoided.
portion of an analytical process. Matrix spiking is a process for
5.5 It is often tacitly assumed that an analyte component is
accomplishing this. The precision and bias estimates from
recovered from samples to approximately the same extent that
several trials under specific analytical conditions represent the
a spike of the same analyte is recovered from a spiked sample.
measurement efficiency with which the analyte may be deter-
One reason that this assumption may be incorrect is that the
mined under these conditions.
spike may not be bound up in the sample (for example, with
3.2.3 spiking solution—the solution in which one or more
suspended matter) in the same way that the naturally occurring
spikes are dissolved (along with any necessary preservatives).
analyte is bound in the sample. The spike may therefore be
This solution acts as a carrier to provide ease of measurement
recovered from the sample differently than the background
and more rapid and thorough mixing of the spike into the
level of the analyte. It is not good practice to correct analytical
sample, as compared to adding the spike as a pure compound.
data using spike recoveries for this reason, as well as the fact
4. Summary of Guide that bias corrections can add variability. However, spike
recovery information should be reported along with related
4.1 This guide describes a technique for the addition of a
sample analysis results.
known amount of an analyte to an aqueous sample. Appropri-
5.6 This guide is also applicable to the use of spikes for
ate concentrations of the spike relative to the original concen-
quantification by the method of standard additions and to the
tration in the sample are discussed. Applications of the tech-
addition of surrogates and internal standards.
nique and aids in the interpretation of results obtained are
described. 6. Apparatus
5. Significance and Use 6.1 Pipetters—Plunger-actuated pipetters, to dispense small
5.1 Matrix spiking is commonly used to determine the bias volumes of spike solutions. These must be calibrated and tested
under specific analytical conditions, or the applicability of a carefully for repeatability before use.
test method to a particular sample matrix in that context, by 6.2 Volumetric Transfer Pipets—Class A, used to deliver
determining the extent to which the spiked analyte or compo- known volumes of sample and to add larger volumes of spiking
nent is recovered from the sample matrix under these condi- solutions.
tions. Reactions or interactions of the analyte or component of 6.3 Volumetric Flasks—Class A volumetric flasks may be
interest with the sample matrix may cause a significant positive used to measure known volumes of sample.
or negative effect on recovery and may render the chosen 6.4 Balance—An analytical (0.1-mg), semimicro (0.01-
analytical, or monitoring, process ineffectual for that sample mg), or micro (0.001-mg) balance.
matrix.
5.2 Matrix spiking can also be used to monitor the perfor- 7. Reagents
mance of a laboratory, individual instrument, or analyst as part 7.1 Purity of Reagents—At a minimum, reagent grade
of a regular quality assurance program. Changes in spike chemicals shall be used in all spike preparations. Reagents of
recoveries or recovery limits from the same or similar matrices the highest available purity shall be used for spike analytes and
over time may indicate variations in the quality of analytical demonstrated to be free of interfering substances for the
results. subsequent tests to be performed. If possible, a primary
5.3 Spiking can be used to compare the recoveries of like standard grade shall be used. Unless otherwise indicated, it is
spikes from reagent water samples and natural matrix samples intended that all reagents conform to the specifications of the
(measured with and without spike) to distinguish between (1) Committee on Analytical Reagents of the American Chemical
unusual interference and (2) inherent method recovery and Society.4 Other grades may be used, provided that the reagent
instability effects. This guide does not attempt to deal with the is of sufficiently high purity to permit its use without adversely
statistical significance of differences in spike recoveries from
different matrices.
5.4 Special precautions shall be observed when nonlabora- 4
Reagent Chemicals, American Chemical Society Specifications, American
tory personnel perform spiking in the field. It is recommended Chemical Society, Washington, DC. For suggestions on the testing of reagents not
listed by the American Chemical Society, see Analar Standards for Laboratory
that all spike preparation work be performed in a laboratory by Chemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeia
experienced analysts so that the field operation consists solely and National Formulary, U.S. Pharmaceutical Convention, Inc. (USPC), Rockville,
of adding a prepared spiking solution to the sample matrix. MD.

2
 D 5810 – 96 (2001)
affecting the bias and precision of subsequent determinations. 9.2 Perform an analysis on at least one portion of the sample
Purchased spiking solutions shall be demonstrated to be free of to estimate the concentration of the component(s) of interest.
substances that would interfere with subsequent analyses being 9.3 Use the result of this analysis to determine the appro-
performed, and the supplier’s stated concentration shall be priate amount of spike and spiking solution to be added to the
verified by analysis prior to use. Compensatory errors associ- sample. If this is not possible (such as when spiking in the
ated with self-referencing should be prevented by using spiking field), estimate the concentrations of the components of interest
solutions of a standard originating from a source, when based on prior knowledge of the sample source.
available, different from that of the routine method calibration
9.3.1 To be of maximum value for quantification of the
standards.
analyte(s) or for the evaluation of method accuracy, the
7.2 Purity of Water—Unless otherwise indicated, references
concentration in the spiked sample should be at least double,
to water shall be understood to mean reagent water as defined
but ideally not over five times, the concentration of the analyte
by the individual test method to be used to analyze a sample
in the unspiked sample, as long as the total analyte concentra-
after spiking. If more than one test method is to be used, the
tion can be brought within the test method’s dynamic range.
minimum criteria of each test method must be met. If test
method reagent water specifications are not available, refer- Spike concentrations below this range lead to highly variable
ences to water shall be understood to mean reagent water as spike recoveries, as described in Section 11. Higher spike
defined by Type I of Specification D 1193 and demonstrated to concentrations may mask the effect that real interferences, such
be free of interfering substances for the test(s) being per- as matrix effects, are having on the analyte at its background
formed. levels, leading to over-optimistic estimates of analyte recovery.
7.3 Solvents—Spectroscopic, high-pressure liquid chroma- 9.3.2 If the spiked component is not present in the sample,
tography (HPLC), or ultrapure grade methanol is preferable for but is added only to validate the recovery of an analytical
use as a solvent for relatively water-insoluble components in method, the concentration after spiking should be at least five
most trace-organic analyses. Other water-soluble solvents may times the detection limit of the method or a concentration of
be useful as solvents for certain analytes. Most inorganic interest to the data user, whichever is greater.
spiking solutions are prepared in water or dilute aqueous acid 9.4 Determine the volume of the portion of sample to be
solution. Solvents shall be checked before use by analysis for spiked, depending on such factors as the sample volume
interfering substances. required by the analytical method to be used, convenience of
7.4 Spiking Solutions—Spiking solutions of each analyte of dilution factors, and amount of sample available.
interest are prepared individually or in combination, either 9.5 Prepare a spiking solution of suitable concentration
gravimetrically or volumetrically. The preservation and storage using the appropriate solvent as described in 7.4.
criteria found in the applicable analytical test method for its
9.5.1 Pertinent factors in determining the appropriate con-
calibration or check standards apply likewise to spiking solu-
centration of the spiking solution are as follows:
tions. The stability of a stored spiking solution should be
verified routinely by the appropriate dilution of a portion of 9.5.1.1 The desired final concentration of the spike in the
spiking solution to the laboratory’s analyte concentration of sample;
interest. Stability is demonstrated whenever the analyzed 9.5.1.2 The working calibration range of the test method for
concentration of a diluted spiking solution falls within the the analyte of interest (the total of the analyte already present
control limits for a routine laboratory control sample of the in the sample and the spiked amount shall fall within this range
same concentration. Where solubilities permit, stock spiking to obtain a useful result);
solutions may be prepared 25 to 100 times as concentrated as 9.5.1.3 The solubility of the solute (the spiked analyte or
the working spike solution and diluted volumetrically to component) in the solvent (water or a water-soluble carrier) of
produce the working spike solution at the time of use. In some the spiking solution;
cases, concentrated solutions may be stable for substantially 9.5.1.4 The volume of the sample; and
longer periods than dilute solutions. Alternatively, prepare
9.5.1.5 The volume markings on the available pipets or
spike or spiking solution fresh for each batch of samples.
pipettors.
8. Sampling 9.5.2 The spiking solution will generally constitute less
8.1 Although sampling methodology is beyond the scope of than, preferably much less than, 2 % of the total volume of the
this guide, a properly split or duplicate sample is of utmost sample, so the matrix is not altered appreciably, for example,
importance to the successful measurement of spike recovery. through matrix solubilizing by the spiking solution carrier
This is especially critical in samples containing suspended solvent. Also, the carrier solvent must not interfere in the test
sediment or volatile components. method. For example, 1 to 2 µL of methanol, a common
8.2 Sample containers shall be selected and prepared, and spiking solution solvent for purge and trap volatile organic
samples shall be preserved in accordance with Practices analytes, in the standard 5-mL sample test portion used will
D 3694. cause false negatives for some ion-trap systems in the area in
which methanol elutes. Less than 0.02 % of the total volume of
9. Procedure the sample should be used in this case. The calculation and
9.1 Use relevant good laboratory practices in accordance correction of the volume of a spiking solution has no negative
with Guide D 3856 and Practice E 200. effect and may be beneficial in any case; see 10.2 and 10.3.

3
 D 5810 – 96 (2001)
9.5.3 The spiking solution volume must be sufficient for the M
P 5 100 T (2)
spiked analyte(s) to remain solubilized and for accurate volu-
metric dispensing. Solubility and handling considerations may
require that the spike be added to the sample as a pure As a practical matter, an analyst may wish to use concen-
compound. Extra care is needed to ensure thorough mixing of tration determinations to calculate P. Readily determined
pure compounds when used as spikes. concentrations and volumes (or masses) may be substituted as
9.6 Add the desired volume or mass of spiking solution or shown in the following two paragraphs. Note that dilution of
spike to the sample. Cap the sample and mix well. the sample by the spiking solution and compensation for
9.7 Examine the spiked sample for any increased turbidity. background levels of the analyte in the sample are considered.
If turbidity persists after extensive mixing, it may be necessary 10.3.1 Assuming that Vs and V are additive (that the final
to spike a new portion of sample using a lower concentration volume of the spiked sample is Vs + V), then A
of component, a smaller volume of more concentrated spiking (Vs + V) − (B 3 Vs) is substituted for M for each analyte and
solution, or a new spiking solution prepared in a more miscible C 3 V is substituted for T. The percent recovery, P, is then
solvent. calculated as follows:
100 [A~Vs 1 V! 2 ~B 3 Vs!#
10. Calculation P5 C3V (3)
10.1 In the following discussion, units of measure are not
given but shall be consistent. That is, the user shall determine where A is the concentration determined by analysis of the
the appropriate concentration units on a case-by-case basis, for analyte in the spiked sample.
example, percent (weight/volume), milligram per litre, or 10.3.2 Where Vs and V are not additive, for example, when
microgram per litre. The units of measure must remain the spiking solution solute is methanol, then, instead of A
consistent throughout these calculations once chosen. For (Vs + V), use the mass, Ms, of the analyte determined by
example, if microgram per litre is selected as the concentration analysis of the spiked sample in the following equation:
units, microgram per litre shall be used wherever concentration
is indicated, litre shall be used wherever volume is indicated, 100 [Ms 2 ~B 3 Vs!#
P5 C3V (4)
and microgram shall be used wherever mass is indicated.
10.2 An estimate of the volume of the spiking solution, V, to
be added to the sample may be calculated as follows: 10.4 Since both A and B are determined experimentally, the
F 3 B 3 Vs acceptable recovery for any spike is a function of the combined
V5 C (1) error in determining both A and B and the relative standard
deviation (RSD) of the method at those concentrations. The
combined error (CE) is determined using the following for-
where: mula:
F = desired ratio of the mass of the analyte added in the
spike to the background mass of the analyte in the CE 5 =~A 3 RSD! 2 1 ~B 3 RSD! 2 (5)
unspiked sample. The value of F should lie between
1 and 4; see 9.3.1. If B is at or below the limit of where:
detectability for subsequent testing, F should equal 4 CE = combined error in the same concentration units as
and B set at the limit of detectability; see 9.3.2, A and B, and
B = measured background concentration of analyte (or RSD = relative overall standard deviation of the test at the
component of interest) in unspiked sample (in vol- concentrations found, expressed as a ratio in ac-
ume, Vs), cordance with Terminology D 4375 assuming the
Vs = volume of sample test portion to which spike is RSD is constant (to sufficient significant figures)
added (with background concentration, B), and over the range of concentrations of A and B. If not,
C = concentration of analyte (or component of interest) in then use RSDA, the RSD at A, at the first occurrence
spiking solution (in volume, V), known by weights in the equation, and RSDB, the RSD at B, at the
and measures from preparation. second occurrence.
It may be observed from Eq 1 that if V becomes large
10.5 Initial Approach to Assessing P—When the percent
relative to V + Vs as explained in 9.5.2, then either increase C
spike recovery, Pi, falls within the generic limits, as follows:
(but not beyond the limits of solubility) or choose an alterna-
tive sample with a smaller background concentration, B, for
testing.
S P̄A 2
3 3 100 3 CE 3 ~Vs 1 V!
C3V # Pi# D (6)

10.3 The percent recovery, P, of the spike is always ex-

pressed as a percentage and is generally calculated from the
ratio of the measured amount (mass), M, of the matrix spike
S P̄A 1
3 3 100 3 CE 3 ~Vs 1 V!
C3V D
found through analysis in the spiked sample to the theoretical
amount (mass), T, of the matrix spike calculated by weights where P̄A is the mean percent recovery expected at concen-
and measures during preparation of the spiking solution. This tration A, then the recovery of the spike is “in control” and
can be expressed as follows: there is no evidence of a significant matrix effect. See ASTM

4
 D 5810 – 96 (2001)
TABLE 1 Effect of Spike-to-Background Ratio on Variability of recovery is not identical to that of the analytical measurement,
Percent Recovery except when the spike-to-background mass ratio is very large.
Spike-to-Background 95 % Tolerance Interval in 11.2 Results outside of three standard deviations about the
Ratio A Expected % Recovery B
mean of historical percent recoveries should be investigated.
100 80 to 120 First consider matrix effects. Bias will typically be in the same
50 80 to 120
10 78 to 122 direction for a given matrix each time that matrix is encoun-
5 76 to 124 tered and will appear only for the problem matrix or matrices.
1 55 to 145
0.5 28 to 170
To obtain a valid result for the sample and to confirm the
0.1 −200 to 400 matrix interference, the analyst should choose another test
0.05 −480 to 680 method or invoke other procedures for resolution, for example,
A
This is F as defined in 10.2. the method of standard additions or the method of standard
B
Based on a relative standard deviation of 10 % in each individual determina- dilutions.
tion, assuming 100 % recovery. Statistical results, including negative recovery
values, are included for reference and are not intended to suggest that actual 11.3 When compared to existing control limits (see 10.6),
negative recovery values are expected. percent recoveries that show a trend, or that are often outside
P̄ 6 3 sP for different matrices, may indicate a bias in the
measurement system. Typically, the analytical instrument (if
MNL 7 5 for an explanation of “in control” and how the there is one involved) or the analyst is causative. Investigation
variability in values of P within these generic limits may be should proceed accordingly. Other sources of out-of-control-
attributable to chance. limit results are possible, for example, a difficult matrix or an
10.6 Control Charting Approach to Assessing P—An ana- interference. If, as recommended in this guide, the spike
lyst or laboratory may accumulate percent recovery values, originates from a source different from the calibration stan-
Pi,i = 1 to n, and, after accumulating at least 8, but preferably 15 dards used for the test method, out-of-specification standards
(or more) Pi values, calculate the mean of the percent recovery, could explain the observed out-of-control-limit results and
P̄, for the number of results available, n, and the standard should be investigated further.
deviation of the Pi, or sP value, using n−1 df for the sample of 11.4 If possible, the standard deviation of the percent
percent recoveries included in these calculations. Based on recovery, sP, determined should be compared with other
Terminology D 4375, these calculations are as follows: analysts, instrument systems, and laboratories running the
n
P same or similar matrices. If the sP value is significantly larger
P̄ 5 ( i
i51 n
(7) than those of other analysts, instruments, or laboratories, the
measurement system should be investigated for the cause.

Œ(
However, note that sP can be quite variable when based on
~Pi 2 P̄! 2
n
sP 5 (8) small numbers of measurements, n. To overcome this limita-
i51 n21
tion, it is suggested that a history of results, Pi,i = 1 to n, be
accumulated (see 10.6) for both analysts, for both instruments,
The recommended control limits are P̄ plus and minus three or in both laboratories, until n is large for each alternative.
times the sP value, and this can be expressed as P̄ 6 3 sP. These Then the two sets of accumulated results are compared before
are control limits for individual future percent recovery values, conclusions are drawn.
P. It may be necessary to segregate concentration or matrix 11.5 If all or most sPvalues exceed, for example, 7 %
categories to optimize control limits. For control charting of P (chosen according to the data quality objectives, 7 % is a
and alternative statistical procedures for calculating control reasonable objective for most quantitative water test methods);
limits, see ASTM MNL 7. 5 that is, if the P̄ 6 3 sP value is outside the range of 80 to 120 %,
the test method variability is considered excessive for the
11. Interpretation of Results analytes and matrices tested, and corrective action should be
11.1 Spike recovery is dependent on the test method used taken, for example, to retrain the analyst, repair or replace the
for analysis, sample matrix, and concentration of the compo- instrument, or find a better test method. See the applicable test
nent of interest in the spiked and unspiked sample. 6 Table 1 method for exceptions and alternative guidance for individual
illustrates the effect of various spike-to-background ratios on analytes. Note that the suggested 80 to 120 % range may be too
the 95 % tolerance interval when the mean recovery is 100 % narrow if, for example, background analyte levels are high (see
and the relative standard deviation of each analytical measure- 11.1) or when working at the extreme lower levels of test
ment is 10 %. It is clear that the variability of the percent method sensitivities, for example, below five times the lower
limit of detection (see 9.3).
12. Keywords
5
ASTM Manual on Presentation of Data and Control Chart Analysis, ASTM
MNL 7, Sixth Edition, ASTM, Philadelphia, PA, 1990.
12.1 bias; internal standards; matrix spike; percent recov-
6
Provost, L. P., and Elder, R. S., “Interpretation of Percent Recovery Data,” ery; quality assurance; recovery; spike; spiking; standard
American Laboratory, Vol 15, No. 12, December 1983, pp. 57–63. additions; surrogates

5
 D 5810 – 96 (2001)

ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentioned
in this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the risk
of infringement of such rights, are entirely their own responsibility.

This standard is subject to revision at any time by the responsible technical committee and must be reviewed every five years and
if not revised, either reapproved or withdrawn. Your comments are invited either for revision of this standard or for additional standards
and should be addressed to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of the
responsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you should
make your views known to the ASTM Committee on Standards, at the address shown below.

This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,
United States. Individual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the above
address or at 610-832-9585 (phone), 610-832-9555 (fax), or [email protected] (e-mail); or through the ASTM website
(www.astm.org).