Thermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

Document au thors: Marita Jok inen, Annu Suoniemi

- Kähärä , Sari Hartikainen, Katja Laine Corresponding address: marita.jokinen@thermofisher .com

Version 5 Page 1 of 20 

# A PPLICATION 

# N OTE 

# :

Drinking Water Orthophosphate for Thermo

Scientific Gallery discrete analyzer

Name of the method : Drinking Water Orthophosphate

Reference : Standard Meth ods (SM) 4500

- P E. Phosphorous / Ascorbic Acid Method.

EPA a pproved method under 40 C

. F

. R . § 141 .23 ( National Primary

Dr inking Water Regulations, NPDWR, based on SDWA , Safe Drinking

Water Act ). 1

Intended use : This paper presents Drinking Water Orthophosphate method for Thermo

Scientific GalleryTM discrete analyzer

. The method was designed to

follow the standard method SM 4500

- P E

. as closely as possible.

Revision number: 5

Revision date (mm/dd/yyyy) : 02/18 /2016



## 1. Scope and Application

1.1 This automated method cover s the determination of ortho phosphate (CAS: 14265

- 44

- 2) in drinking water with the Thermo ScientificTM Gallery TM discrete analyzer

.

1.2 The method is based on reaction that is speci fic for the ortho phosphate ion.

1.3 The applicable range for this method is from 0.01 2 5

– 0.5 mg PO 4

- P/L. An ext ension of the range to 5 mg PO 4

- P/L is achieved when 1:10 automated dilution is configured. The automated dilution feature must be confirmed with acceptable analysis of quality control samples by the user

.



## 2. Summary of Method

2.1 Ammonium molybdate and antimony potassium tartrate react in an acidic medium with dilute solutions of phosphorous to form an antimony

- phospho

- molybdate complex. This complex is reduced to an intensely blue

- colored complex by ascorbic acid. The color is proportional to the orthophosphate concentration.

2.2 Only orthophosphate forms a blue color in the test. Poly phosphate s (and some organic phosphate compounds) may be converted to the ortho phosphate form by sulfuric acid hydrolysis. Organic phosphate compounds may be converted to the ortho phosp hate form by persulfate digestion. These conversion methods are not covered in this Application Note.Thermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

Document au thors: Marita Jok inen, Annu Suoniemi

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Version 5 Page 2 of 20 

## 3. Definitions

3.1 Units and symbols from the international metric system (SI) are used. Definitions, acronyms,

and abbreviati ons are explained as they occur fo r the first time.



## 4. Interferences

4.1 No interference is caused by copper, iron or silicate at concentrations many times greater than their reported concentration in seawater. However, high iron concentrations can cause precipitation of, and subsequent loss, of phosphorous.

4.2 Arsenate s react with the molybdate reagent to produce a blue color similar to that formed

with orthophosphate

. Thus, it should be considered when present in concen trations higher

than orthophosphate . C oncentrations as low as 0.1 mg a s/L can in terfere with the orthophosphate determination.

4.3 Hexavalent chromium and nitrite

- ion can interfere with the orthophosphate determination yielding in approximately 3 % lower results at concentrations of 1 mg/L and 10 % to 15 %

lower re sults at 10 mg/L concent rations

.

4.4 Sample turbidity is not to be removed by filtration prior to analysis for ortho phosphate

, unless dissolved reactive phosphorous is studied. Samples for total phosphorous may be filtered only after digestion. Sample color that absorbs in the photom etric range used for analysis will also interfere. The sample blank is measured automatically in the application for sample color and turbidity correction.



## 5. Safety

5.1 The toxicity and carcinogenicity of each reagent used in this method have not been fully

est ablished. Each chemical should be regarded as a potential health hazard and exposure should be as low as reasonably achievable

.

5.2 The following chemicals have the potential to be toxic or hazardous. C onsult MSDS (Material Safety Data Sheet) for details.

5.2.1 Sulf

uric acid (contained in Phosphate R1) (CAS # 7664

- 93

- 9)

5.2.2 Ammonium molybdate tetrahydrate (contained in Phosphate R1) (CAS# 12027

- 67

- 7)

5.2.3 Antimony potassium tartrate (contained in Phosphate R1) (CAS# 28300

- 74

- 5)

5.2.4 Ascorbic acid (contained in Phosphate R2) (CAS# 5 0

- 81

- 7)

5.2.5 Potassium phosphate monobasic (CAS# 7778

- 77

- 0)Thermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 3 of 20 

## 6. Equipment and Supplies

6.1 Balance: Analytical, capable of accurately weighing to the nearest 0.0001 g.

6.2 Water purification system for producing suitable water for autoanalyzers . Refer to instrument

user manual.

6.3 Thermo Scientific Gallery , automated discrete photometric system

.

6.4 Filter, wavelength 880 nm.

6.5 DECACELL TM Cuvettes for Thermo Scientific Gallery . DECACELL cuvettes must always

be use d with Thermo Scientific Gallery . Cuvettes are for single use only.

6.6 Washi ng solution 4.5 % hypochlorite solution, 4 × 20 mL, is used for daily instrument cleansing.



## 7. Reagents and Standards

Reagents for Gallery: THERMO SCIENTIFIC ORDERING NUMBERS

984366 Phosphate R1, 4 × 20 mL

984367 Phosphate R1, 20 × 20 mL

984368 P hosphate R2, 4 × 20 mL

Standard s for Gallery: THERMO SCIENTIFIC ORDERING NUMBER

984729 Phosphate (as P ) Std

, 1000 ppm, 500 mL (calibration)

984726 Phosphate (as P O4 ) Std

, 1000 ppm, 500 mL ( QCS )

7.1 Preparation of reagents needed in this method is describe d under. Also ready to use reagents

are available for this method.

7.2 Reagent water

— Distilled or deionized water, free of the analyte of interest . Water stored in bottles should be substituted by fresh water after one week.

7.3 Phosphate R1

— Prepared by adding 15 mL of a mmonium molybdate solution (2 0 g (NH 4 ) 6 Mo 7 O 24 ∙4H 2 O /500 mL water) to 50 m L of s ulfuric acid solution (70 mL conc. H 2 SO 4 /500 mL water) followed by 5 mL addition of a ntimony potassium tartrate solution (1.5 g K(SbO)C 4 H 4 O 6 ∙1⁄2H 2 O /5 00 mL water) to this mixture.

7.4 Phosphate R2

— Ascorbic acid solution cont aining 1.76 g ascorbic acid in 100 mL water. This solution is stable for 5 days when refrigerated between 2

- 8 °C.

7.5 Phosphate (as P ) Std

, 1000 ppm

— Phosphate standard 1000 mg /L as P is for calibrating the Gallery instrument.

7.6 Phosphate calibration solution s

— Prepare an appropriate series of standards by diluting

suitable volume s of Phosphate standa rd (stock) with distilled water, or use automated

Galler y calibration dilution feature

.

7.7 Quality Control Solution (QCS)

— A second source standard from an externa l source, e.g. Phosphate (as P O4 ) Std , 1000 ppm (326.2 ppm as P)

. Dilute to appropriate concentration

with distilled water. Do not use Phosphate calibration solutions as QCS

- samples.Thermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

Document au thors: Marita Jok inen, Annu Suoniemi

- Kähärä , Sari Hartikainen, Katja Laine Corresponding address: marita.jokinen@thermofisher .com

Version 5 Page 4 of 20 7.8 Laboratory Fortified Blank (L FB )

— Phosphate standard (stock) 1000 mg/L or self made

standard solution. Dissolve 0.4394 g of predried (105 °C for one hour) p otassium phosphate monobasic (KH 2 PO 4 ) in distilled water and dilute to 1 000 mL. 1 .0 mL of this solution contains 0.1 mg P. Dilute to appropriate concentration with distill ed water. Do not use Phosphate calibration solutions as LFB

- samples.



## 8. Sample Collection, Preservation, and Storage

8.1 Sample containers may be of plast ic material or of Pyrex glass. All bottles must be thoroughly cleaned and rinsed with distilled water. Volum e collected should be sufficient to insure a representative sample, allow for replicate analysis (if required), and minimize waste disposal. Do not store samples containing low concentrations of phosphorous in plastic

bottles because orthophosphate s may be adsorbed to the walls of plastic bottles.

8.2 Samples should be analyzed as soon as possible

, after collection , within 48 hours at

maximum . Samples must be cooled and maintained at 4 °C

.



## 9. Quality Control

9.1 Each laboratory using this method is required to operat e a formal quality control (QC) program. The minimum requirements of this program consist of an initial demons tration of laboratory capability and the periodic analysis of laboratory reagent blanks, continuing

calibration c heck standards, fortified blanks and fortified samples as duplicates as a continuing check on performance. The laboratory is required to maintain performance records that define the quality of the data that are generated.

9.2 The initial demonstration of capability (IDC) is used to characteri ze instrument performance

in a n individual laboratory by determination of calibration curve and analysis of quality control samples ( QCS) and laboratory performance by determination of method detection limit (MDL)

, minimum reporting level (MRL) and the ini tial precision and recovery (IPR)

test.

9.2.1 Calibration C urve

— C alibration curve must be determined initially and reanalyzed at
least every six months or whenever a significant change in instrument response is observed or expected. The calibration curve must use a minimum of five standards and a blank to ensure that the resulting curve is fitted correctly. The Gallery analyzer fits the calibration first as a linear curve. The user can then fit the calibration to 2nd degree curve, if the fitting is better ther eby. The correlation coefficient of the calibration curve

should be equal to or greater than 0. 995

.

9.2.2 Quality Control Sample ( QCS )

— When beginning the use of this method, whenever

new standard materials are used

, on a quarterly basis or as required to meet data

- quality needs, verify the calibration standards and acceptable instrument performance with the analyses of a secondary standard solution (QCS) from an external source . If the

determined concentrations are not within ± 1 5 % or ± 20 % when the concentr ation is ≤

2x MRL of the stated values, performance of the determinative step of the method isThermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 5 of 20

unacceptable. The source of the problem must be identified and corrected before proceeding with t he initial determination of MDL or continuing with on

- going anal yses

.

9.2.3 Method Detection Limit (MDL )

— An MDL should be established using reagent water (blank) fortified at a concentration of one to five times the estimated instrument

detection limit (MDLest)

. The estimate is calculated as three times the standard

deviat ion of replicate instrumental measurements of the analyte in reagent water.

MDL est = 3 × (S 0 )

where S 0 = standard deviation of the replicate analyses (n=10) of reagent

water.

P repare a laboratory fortified blank sample (LFB) at a concentration which is at least equal to or in the same concentration range as the esti mated method detection limit (r ecommend ed 1

– 5 x MDLest )

. Perform all calculations defined in the method and report the concentration values in the appropriate units. Each individual resu lt must be

within 70

– 130 % of the theoretical value. Calculate the MDL as follows:

MDL = (t) × (S)

where t = Student's t

- value for a 99 % confidence level and a standard

deviation estimate with n

- 1 degrees of freedom [t = 3.14 for 7 replicat es ]

S = standard deviation of the replicate analyses

MDL should be determined every six months, when a new operator begins work, or whenever there is a significant change in the background or instrument response.

9.2.4 Minimum Reporting Level (MRL) Confirmation

— The minimum concentration that can be reported by a laboratory as a quantified value for the method analyte in a sample following analysis. The MRL must be at or above the level of the lowest fortified

calibrator , where it must meet the criteria set for MRL confirmation. It would also have

to be considered that criteria for Laboratory Reagent Blank (LRB) must be met (LRB ≤

1/3 x MRL ).

Fortify and analyze seven replicate LFBs at the proposed MRL concentration. Calculate the mean ( Mean ) and standard deviat ion (s) for these replicates. Determine the Half Range for the Prediction Interval of Results (HR PIR ) using the equation:

HR PIR = 3.963s

Where s = standard deviation

3.963 = constant value for seven replicates

Calculate the upper and lower limit s f or the Prediction Interval of Results (PIR = Mean

+HR PIR ) from the results and confirm that the results meet the criteria. Accepted results

confirm the MRL to be validated.Thermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 6 of 20

Criteria: The Uppe r PIR Limit m ust be ≤ 150 % recovery. The Lower PIR Limit must be ≥ 50 % recovery

9.2.5 Initial P recision and R ecovery (IPR)

— For initial precision and recovery test the laboratory should analyze four replicate volumes of reagent water spiked with the analyte of int e rest (LFB) . Calculate accuracy as percent rec overy and precision as relative standard deviation (% RSD) as shown under.

where = mean of replicate measurements

= measured value of the rep licate n = number of replicates

where Cs = spiked sample concentration

C = sample background concentration s = concentration equivalent of analyte added to sample

Criteria for % R ecovery: ±15% (or 85

- 115 %), when c is > 2 x MRL and ±20% (or 80

- 120 %), when c is ≤ 2 x MRL

Criteria for % RSD: ±15 %

9.3 Asse ssing laboratory performance with ongoing QC includes the use of Laboratory Reagent Blank (LRB)

, Laboratory Fortified Blank (LFB) , Continuing Calibration Check (CC C)

9.3.1 Laboratory Reagent Blank (LRB)

— The laboratory should analyze at least one LRB with each batch of samples. Data produced are used to assess contamination from the laboratory environment.

Criteria for LFB : Must be below 1/3 of MRL

9.3.2 Laboratory Fortified Blank (LFB)

— The laboratory should analyze at least one LFB with each batch of samples. Calculate accuracy as percent recovery as shown in 9.2.5 . IfThermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 7 of 20

the recovery of any analyte falls outside the required control limits

, the source of the problem should b e identified and resolved before continuing analyses. LFB analyses data is used to assess laboratory performance agai nst the required control limits.

Criteria for % recovery: ±15% (or 85

- 115 %), when c is > 2 x MRL and ±20% (or 80

- 120 %), when c is ≤ 2 x MRL

When enough data from LFB samples analyzed as part of method ongoing QC is gathered the laboratory can assign its own control limits from the standard deviation of

the results.

9.3.3 Continuing Calibration Check (CC C)

— For all determinations th e laboratory should analyze the CCC (a mid

- range check standard) and a calibration blank immediately following calibration , after every tenth sample and at the beginning and end of the sample run

. Analysis of the CCC solution and calibration blank immediat ely following calibration must verify that the instrument is within ± 10 % of calibration. Subsequent analyses of the CCC solution must verify the calibration is still within ± 10 %. This procedure is done automatically by the analyzer

. Note ! Application h as manual result

acceptance instead of automatic acceptance to ensure CCC protocol is followed.

If the calibration cannot be verified within the limits specified in the application

, the analyzer gives a message of outlier result. In such case reanalyze the CCC solution by triggering the Ongoing QC procedure . If the second analysis of the CCC solution confirms calibration to be outside the limits, sample analysis must be discontinued, the

cause determined and/or in the case of drift the instrument recalibrat ed. All samples following the last acceptable CCC solution must be reanalyzed, even if the resumed

CCC is acceptable. The analysis data of the calibration blank and CCC solution must be kept on file with the sample analyses data.

Criteria: CCC % recovery ±10% ( or 90

- 110 %)

9.3.4 Quality Control Samples (QCS)

— S tandard solution sample s (9.2.2) , that must reach laboratory acceptance criteria are analyzed with each calibration, whenever new standard materials are used, on a quarterly basis or as required to me et data

- quality

needs in addition to the IDC measurements.

Criteria: ±15% (or 85

- 115 %), when c is > 2 x MRL and

±20% (or 80

- 120 %), when c is ≤ 2 x MRL

9.4 Laboratory Fortified Matrix Sample

9.4.1 Laboratory Fortified Matrix Sample (LFM) (also termed Matri x Spike, MS and Matrix Spike Duplicate MSD )

— The laboratory must add a known amount of analyte to a minimum of 10 % of the routine samples. The analyte concentration must be high enough to be detected above the original sample and should not be less than four timesThermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 8 of 20

the MDL. The added analyte concentration should be the same as that used in the laboratory fortified blank in section 9.3.2

.

Calculate the percent recovery for each analyte, corrected for concentrations measured in the unfortified sample, and co mpare these values to the designated LFM recovery range 85

- 115 % . If the recovery of any analyte falls outside the designated LFM recovery range and the laboratory performance for that analyte is shown to be in control, the recovery problem encountered wit h the LFM is judged to be either matrix or solution related, not system related. The Gallery analyzer has a standard addition feature for difficult sample matrixes.

where C

s = spiked sample concentration

f = spike dilution correc tion C = sample background concentration s = concentration equivalent of analyte added to sample

Note! If the added spike volume is less than 1% of the total LFM sample volume, the

factor f can be excluded.

Criteria: LFM % Recovery: ±15%

9.4.2 Laboratory Fortified Matrix Sample (LFM) Precision

— The precision of the LFM determinations is also assessed by measuring LFM samples in three replicates followed by calculation of relative standard deviation ( % RSD ) of the results as shown in 9.2.5

.

In routine la boratory performance evaluation LFM duplicate samples (LFMD, also

termed Matrix Spike Duplicate or MSD) are used. Precision is then calculated as

follows:

where LFM = analyte concentration measured i n LFM sample

LFMD = analyte co ncentration measured in LFM duplicate

Criteri a: % RSD ±20%



## 10. Calibration and Standardization

10.1 Dilute Phosphate standard (stock) ( Thermo Scientific ) with distilled water to get a suitable phosphate standard for calibration

. Use the automated Gallery calibrat ion dilution feature for a calibration curve. Alternatively, p repare a series of at least five standards, covering the desired range, and a blank, for a calibration curve.

10.2 Process standards and blan ks as described in Section 11.0 Procedure.Thermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 9 of 20 10.3 The Gallery an alyzer plots automatically instrument response against standard concentration. The user must accept this calibration curve before the analyzer starts to measure blanks and samples. The calibration correlation coefficient shall b e equal to or greater than 0 .995

.

10.4 After the calibration has been established, it must be verified by the analysis of a suitable control sample (QCS) . If measurements exceed ± 15 % of the established LFB value, the analysis should be terminated and the instrument recalibrated. Period ic reanalysis of the QCS

is r ecommended quarterly at minimum. Ongoing QC is done automatically by the analyzer and it includes analyzing LRB, CCC and LFB samples with each batch of ten samples. In addition LFM samples are to be done in duplicates with ever y batch of ten samples.



## 11. Procedure

11.1 Preparation before analysis

— Add all required reagents, samples

, o ther consumables and requests for tests follow ing the instrument instructions according to the manufacturer

.

11.2 Gallery Drinking Water Orthophosphate test f low

— To 120 μL of sample or standard or

blank 14 μ L of Phosphate R1 (Thermo Scientific) is added and solution is mixed. After blank

measurement

, 6 μ L of Phosphate R2 (Thermo Scientific) is added and solution is mixed. After 9 minutes incubation, the absorb ance is measured at 880 nm. The application is shown in Appendix A

.



## 12. Data Analysis and Calculation

12.1 The Gallery analyzer plots automatically instrument response against standard concentration making a calibration curve. The instrument computes sample concent ration by comparing sample response with the standard curve.

12.2 R esults are reported in mg P/L.



## 13. Method Performance

13.1 According to a validation study of the Gallery Drinking Water Orthophosphate method, single laboratory result for MDL was 0.00036 mg/L as P and MRL was confirmed to be

0.01 25 mg/L as P. Initial precision was analyzed to be 0.1

- 1.0 % RSD and accuracy 102

- 103 % recovery at concentrations 0.1

- 0.4 mg/L as P.

13.2 Precision for LFB samples was 0.2

– 1.1 % RSD and for LFM samples 0.1

– 0.7 % RSD

correspon ding to sample concentrations 0.1

– 0.4 mg/L as P.

13.3 Accuracy for LFB samples was 102

– 103 % recovery and for LFM samples 92

– 98 % recovery corresponding to sample concentrations 0.1

– 0.4 mg/L as P.

13.4 CCC standard recovery was 100

– 105 % and LRB was always below MDL.

13.5 Method limits for LFB and LFM samples are 15 % RSD and 85

- 115 % Recovery and for

CCC 90

- 110 % Recovery . LRB must be below MDL.Thermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 10 of 20 

## 14. Pollution Prevention

14.1 The instrume n t uses small amounts of reagents, which reduces the quantity of wastes signifi cantly compared to manual methods or flow analyzers. The small packing size facilitates the use of reagents during their shelf lives and thus reduces disposal cost of unused

materials.



## 15. Waste Management

15.1 Excess reagents, samples and method process wastes sh ould be characterized and disposed of in an acceptable manner.

15.2 The containers for cuvette and liquid waste must be emptied and rinsed with water at the end of the day.



## 16. References

16.1 4500

- P E. PHOSPHOROUS/Ascorbic acid method. Standard Methods for the Examina t ion of Water and Wastewater, 22nd Edition, American Public Health Association (APHA),

2012

.

16.2 Code of Federal Regulations 40 § 141.23 . Inorganic chemical sampling and analytical requirements

.

16.3 Code of Federal Regulations 40 § 136, Appendix B

— Definition and Procedure for the

Determination of the Method Detection Limit

– Revision 1.11.

16.4 4020 Quality Assurance/Quality Control

. Standard Methods for the Examina tion of Water and Wastewater, 22nd Edition, American Public Health Association (APHA), 2012

.

16.5 1020

- B.4 Me thod Detection Level Determination and Application. Standard Methods for the

Examina ti on of Water and Wastewater, 22nd Edition, American Public Health Association (APHA), 2012

.



## 17. T ables, diagrams, flowcharts and validation data

17.1 Test parameters and test flow for Gallery Drinking Water Orthophosphate method are presented in Appendix A.Thermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

Document au thors: Marita Jok inen, Annu Suoniemi

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Version 5 Page 11 of 20 17.2 Method performance data

17.2.1 Calibration and QCS

- samples

Calibration curve was prepared between 0

- 0.5 mg P/L for DW o

- PO4P application.

Calibration was done with automatic diluti on from working standard solution of 1.5 mg

P /l and laboratory reagent water. Calibration was verified with QCS

- samples.

Figure 1 . Calibration curve for DW o

- PO4P application

.

Table 1

. Results for Phosphate QCS

- samples after cal ibration.

Orthophosphate as phosphorous

QCS

- sample Duplicate % RSD % Recovery
PO4P 0.1mg/l 0.13 % 96 %
PO4P 0.4mg/l 0.19 % 103 %

17.2.2 Method Detection Limit (MDL)

MDL was established using reagent water (blank) fortified at a concentration approximately five times the estimated instrument detection limit (MDLest). The

estimate was calculated as three times the standard deviation of replicate instrumental measurements of the analyte in reagent water.

MDL est = 3 × (S 0 )

where S 0 = standard deviation of t he replicate analyses (n=10) of reagent

water.Thermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 12 of 20

Table 2

. Values for determining MDLest for DW o

- PO4P application

. S ample Result s (mg/L)

LRB m in. 0

. 0018 0 0

LRB m ax. 0.002122

average = 0.00188

n = 10

std. deviation (SD) = 0. 00 013

MDLest calculated = 0

. 000402
MDLest assigned= 0

. 0022

NOTE! The MDL est had to be assigned higher than the calculated MDL est, because the highest individual LRB result of this test was as high as 0. 002122 mg/l as P

, and criteria for LRB sample in t his test is , that it should never be above the MDL est

.

A laboratory fortified blank sample (LFB) at a concentration which was approximately five times the MDLest, was analyzed in seven replicates

. Each individual result had to

be within 70

– 130 % of the theoretical value. MDL w as calculated as follows:

MDL = (t) × (S)

where t = Student's t

- value for a 99 % confidence level and a standard

deviation estimate with n

- 1 degrees of freedom [t = 3.14 for 7 replicates]

S = standard deviation of the r eplicate analyses

Table 3

. Values for determining MDL for DW o

- PO4P application.

Sample Result (mg/L) % Recovery

LFB 0

. 01 mg/l PO4P 0

. 00793 79 %

LFB 0

. 01 mg/l PO4P 0

. 00791 79 %

LFB 0

. 01 mg/l PO4P 0

. 00795 80 %

LFB 0

. 01 mg/l PO4 P 0

. 00768 77 %

LFB 0

. 01 mg/l PO4P 0

. 00796 80 %

LFB 0

. 01 mg/l PO4P 0

. 00803 80 %

LFB 0

. 01 mg/l PO4P 0

. 00797 80 %

average = 0

. 007919 79 %

n = 7

std. deviation (SD) = 0

. 000114

% RSD = 4

. 5 %

MDL = 0

. 00036Thermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 13 of 20 17.2.3 Minimum Reporting L evel ( MRL )

S even repl icate LFBs were analyze d at the proposed MRL concentration.

T he mean ( Mean ) and standard deviation (s) for these replicates were calculated

. The Half Range for the Prediction Interval of Results (HR PIR ) was determined using the equation:

HR PIR = 3.963s

Where s = standard deviation

3.963 = constant value for seven replicates

The upper and lower limits for the Prediction Interval of Results (PIR = Mean +HR PIR )

were calculated from the results.

Crite ria: The Uppe r PIR Limit must be ≤ 150 % recovery.

The Lower PIR Limit must be ≥ 50 % recovery

Table 4 . Results for MRL confirmation for DW o

- PO4P application

. Sample Result (mg/L) % Recovery

LFB 0

. 01 mg/l PO4P 0

. 010088 101 %

LF B 0

. 01 mg/l PO4P 0

. 009663 97 %

LFB 0

. 01 mg/l PO4P 0

. 009568 96 %

LFB 0

. 01 mg/l PO4P 0

. 009378 94 %

LFB 0

. 01 mg/l PO4P 0

. 009891 99 %

LFB 0

. 01 mg/l PO4P 0

. 009616 96 %

LFB 0

. 01 mg/l PO4P 0

. 009605 96 %

average = 0

. 00969 97 %

n = 7

std. deviation (s) = 0

. 000232

HR PIR = 0

. 000921

Upper PIR Limit = 106

. 1 %

Lower PIR Limit = 87

. 7 %

% RSD= 2

. 4 %

Calculated limits met the criteria and confirmed the MRL to be validated.Thermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 14 of 20 17.2.4 Initial Precision and R ecovery (IPR)

F our replicate volumes of reagent water sp iked with the analyte of interest (LFB) were analyzed at three different concentrations. % recovery and % RSD were calculated.

Results are shown in table 7.

Criteria for % recovery: 85 %

- 115 %, when c is > 2 x MRL and

80 %

- 120 % , when c is ≤ 2 x MRL

Criteria for % RSD: ±15%

Table 5

. Results for initial precision and recovery (IPR) for DW o

- PO4P application. Sample Result (mg/L) % Recovery

LFB 0

. 1 mg/l 0

. 1027 103 %

LFB 0

. 1 mg/l 0

. 1028 103 %

LFB 0

. 1 mg/l 0

. 1027 103 %

LFB 0

. 1 mg/l 0

. 1028 103 %

average 0

. 1027 103 %

std.deviation 0

. 000074

% RSD 0

. 1 %
Acceptable YES YES

LFB 0

. 3 mg/l PO4P 0

. 3056 102 %

LFB 0

. 3 mg/l PO4P 0

. 3020 101 %

LFB 0

. 3 mg/l PO4P 0

. 3096 103 %

LFB 0

. 3 mg/l PO4P 0

. 3051 102 %

average 0

. 3056 102 %

std.deviation 0

. 0031

% RSD 1

. 0 %
Acceptable YES YES

LFB 0

. 4 mg/l 0

. 4074 102 %

LFB 0

. 4 mg/l 0

. 4083 102 %

LFB 0

. 4 mg/l 0

. 4088 102 %

LFB 0

. 4 mg/l 0

. 4083 102 %

average 0

. 4082 102 %

std.deviation 0

. 00059

% RSD 0

. 1 %
Acceptable YES YESThermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 15 of 20 17.2.5 Pr ecision and accuracy from LFB

Precision (%RSD) and accuracy (% Recovery) w ere determined using LFB samples at 0.1, 0.3 and 0.4 mg / L concentrations

. Results are shown in table 8.

Criteria for % recovery: 85

- 115 % , when c is > 2 x MRL and

80

- 120 % , when c is ≤ 2 x MRL

Criteria for % RSD: ±15%

Table 6

. Precision and accuracy data for DW o

- PO4P application.

Sample:

Concentration: LFB 0.1 mg/l PO4P 0.1 mg P/L LFB 0.3 mg/l PO4P

0.3 mg P/L LFB 0.4 mg/l PO4P

0.4 mg P/L

average = 0.10257 0.30541 0.40891

n = 10 10 10

std. de v. (SD) = 0.000209 0.003242 0.000767

% RSD = 0.2 % 1.1 % 0.2 %
Avg. % Recovery = 103 % 102 % 102 %

Acceptable YES YES YES

17.2.6 Precision and accuracy from LFM samples

Laboratory Fortified Matrix (LFM) samples were pre pared by spiking three different tap water samples with Phosphate standard solution to gain spike concentrations of 0.1 and 0.4 mg/L as P for each sample

. The analyte concentration s were high enough to be detected above the original sample and were not les s than four times the MDL. The added analyte concentration s were chosen to be the same as those used for LFB. Precision was estimated by analyzing four replicates and calculating the %RSD. Accuracy was estimated from LFM and corresponding unspiked samples by analyzing them and calculating the % Recovery using the next equation. Results for LFM precision and accuracy are shown in table 9.

where C

s = spiked sample concentration

f = spike dilution correction C = sample background c oncentration s = concentration equivalent of analyte added to sample

Note! Because the added spike volume was less than 1% of the total LFM sample

volume (0.4 % and 0.12 % of total LFM sample volume) , the factor f could be excluded.

Criteria for LFM % recovery : 85

- 115 % , when c is > 2 x MRL and

80

- 120 %, when c is ≤ 2 x MRL

Criteria for LFM % RSD: ±15%Thermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 16 of 20

Table 7 . Laboratory Fortified Matrix (LFM) sample results for DW o

- PO4 application.

Sample Result avg. n = 3 (mg/L) Spike.

mg/L % Recovery % RSD.

n =3 Acceptab le

TW

- 02 0.0021

TW

- 02 +0.1 mg/l PO4P 0.0946 0.10 92 % 0.7 % YES

TW

- 02 +0.4 mg/l PO4P 0.3906 0.40 97 % 0.1 % YES

TW

- 03 0.0026

TW

- 03 +0.1 mg/l PO4P 0.0944 0.10 92 % 0.3 % YES

TW

- 03 +0.4 mg/l PO4P 0.3943 0.40 98 % 0.2 % YES

TW

- 04 0.0028

TW

- 04 +0.1mg/l PO4P 0.0989 0.10 96 % 0.7 % YES

TW

- 04 +0.4mg/l PO4P 0.3754 0.40 93 % 0.3 % YES

Average = 95 % 0.4 % YES

17.2.7 Continues Calibration Check (CCC)

Continues Calibration Check (CCC) solution was analyzed in intervals of every 10 th sample, at the beginning and end of run. CCC sample concentration was from the mid

calibration area

– 0.25 mg/L. CCC sample result summary is in table 10.

Criteria for CCC % recovery: 90

– 110 %

Table 8 . PO4P CCC result summary. Sample : PO4P CC C, concentration 0. 25 mg/L

.

Analyzer 1 Analyzer 2
Average: 0

. 254 0

. 256

% Recovery 102 % 102 %

n = 20 6

std. deviation (SD) = 0

. 003 3 0

. 00 48

% RSD= 1

. 29 % 1

. 87 %

Minimum result: 0

. 249 0

. 252

Maximum result: 0

. 261 0

. 261
Acceptable YES YESThermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 17 of 20 17.3 The valid ation study results showed the Drinking Water Orthophosphate method for Thermo Scientific Gallery discrete analyzer is equally effective in meeting the QC acceptance criteria given in the reference method.

Table 9 . Performance of t he Drinking Water Orthophosphate method for Thermo Scientific Gallery

discrete analyzer compared to reference method acceptance criteria.

QC acceptance criteria for SM 4500

- P E Performance of DW o

- PO4P for Gallery analyzer

Minimum detectable concentration : 10 μg

P/l

Recovery IPR: NA (laboratory specific) % Recovery CCC: 90

– 110 % Recovery LFB: Mean ± 3 x SD (n ≥ 20) Recovery LFM: NA (laboratory specific)

% RSD IPR: NA (laboratory specific) % RSD LFB: NA (laboratory specific) % RSD LFM: NA (laboratory sp ecific)

Method Blank: ≤ 1⁄2 x MRL MDL calculated: 0.00036 mg P/L

MRL: 0.0125 mg P/L

% Recovery IPR: 102

– 103% % Recovery CCC: 100

– 105% % Recovery LFB: 102

– 103% % Recovery LFM: 92

– 98 %

% RSD IPR: 0.1

– 1.0 %

% RSD LFB: 0.2

– 1.1 %

% RSD LFM: 0.1

– 0.7 %

Method Blank (LRB): ≤ 1/3 x MRLThermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 18 of 20 

## APPENDIX A

.

Test flow for DW o

- PO4P application in the Gallery analyzer.Thermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

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- Kähärä , Sari Hartikainen, Katja Laine Corresponding address: marita.jokinen@thermofisher .com

Version 5 Page 19 of 20Thermo Fisher Scientific, Analyzers & Automation, R&D Industrial diagnostics

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Version 5 Page 20 of 20