Supporting information Characterization and validation of candidate reference methods for the determination of calcium and magnesium in biological fluids Loai Aljerf a,*, Ammar Mashlah b a
Department of Life Sciences, Faculty of Dentistry, University of Damascus, Damascus, Syria
b
*
Oral Medicine Department, Faculty of Dentistry, University of Damascus, Damascus, Syria
Correspondence to: L. Aljerf, Department of Life Sciences, Faculty of Dentistry, University of Damascus, Damascus, Syria.
Corresponding author. E-mail address:
[email protected] (L. Aljerf).
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1.
Analytical definitions
Analyte, A specific chemical moiety being measured, this can be intact drug, biomolecule or its derivative, metabolite and/or degradation product in a biologic matrix Analytical Procedure, refers to the way of performing the analysis and it should describe in detail the steps necessary to perform each analysis Incurred Sample Reanalysis, is the analysis of a portion of the incurred samples to determine whether the original analytical results are reproducible. Sample Matrix, or Matrix, defines the general physical-chemical makeup of a particular sample Sensitivity, means the ability of a method or instrument to detect an analyte at a specified concentration. (NR 149.03(28m)) Signal-to-Noise Ratio (S/N), is a dimensionless measure of the relative strength of an analytical signal (S) to the average strength of the background instrumental noise (N) for a particular sample and is closely related to the detection level. The ratio is useful for determining the effect of the noise on the relative error of a measurement. The S/N ratio can be measured a variety of ways, but one convenient way to approximate the S/N ratio is to divide the arithmetic mean (average) of a series of replicates by the standard deviation of the replicate results [1] Accuracy, is a combination of the bias and precision of an analytical procedure, which reflects the closeness of a determined value to a true limit. The accuracy is accepted either as a conventional true parameter or an accepted reference factor. For the purposes of laboratory certification, accuracy means the proximity of a measured value to its generally accepted value or its value based upon an accepted reference standard. (NR 149.03(2)) (Standard Methods, 18th edition) Sample Standard Deviation, or Standard Deviation (s), is a measure of the degree of agreement, or precision, among replicate analyses of a sample. In this document, standard deviation implies sample standard deviation (n-1 degrees of freedom). However, the population standard deviation (n degrees of freedom) should only be used when dealing with a true approximation of a population (e.g. greater than 25 data points). (Standard Methods, 18th edition)
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Precision, is a measure of the random error associated with a series of repeated measurements of the same parameter within a sample. Therefore, it describes the closeness of concurrence (degree of scatter) between a series of measurements obtained under the prescribed conditions with which multiple analyses of a given sample agree with each other, and is sometimes referred to as reproducibility. Precision is determined by the absolute standard deviation, relative standard deviation, variance, coefficient of variation, relative percent difference, or the absolute range of a series of measurements. (s. NR 140.05(16) and Standard Methods, 18th edition) Bias, provides a measure of systematic, or determinative error in an analytical method. Bias is determined by assessing the percent recovery of spiked samples. Historically, the term accuracy has been used interchangeably with bias, although many sources make a distinction between the two. (Standard Methods, 18th edition) Linear Calibration Range (LCR), or Range of Linearity, is the region of a calibration curve within which a plot of the concentration of an analyte versus the response of that particular analyte remains linear and the correlation coefficient of the line is approximately 1 (0.995 for most analytes). The plot may be normal-normal, log-normal, or log-log where allowed by the analytical method. At the upper and lower bounds of this region (upper and lower limits of quantitation), the response of the analyte's signal versus concentration deviates from the line Limit of Detection (LOD) or Detection Limit, is the lowest concentration level that can be determined to be statistically different from a blank (99% confidence). The LOD is typically determined to be in the region where the signal to noise ratio (S/N) is greater than 5 or by a statistical approach based on measuring replicate blank (negative) samples. Limits of detection are matrix, method, and analyte specific. The LOD procedure is based upon a method developed by A. Hubaux and G. Vos [2] that takes into account the effects of bias and concentration on the calculated LOD. Their paper gives useful insight for calculating detection limits, and demonstrates the importance of biases at different concentration levels. (ss. NR 140.05(12) & 149.03(15)) Instrument Detection Limit (IDL), is the concentration equivalent to a signal, due to the analyte of interest, which is the smallest signal that can be distinguished from background noise by a particular instrument. The IDL should always be below the method detection limit (MDL), is not used for compliance data reporting, but may be used for statistical data analysis and comparing the attributes of different instruments. The IDL is similar to the "critical level" and "criterion of detection" as defined in the literature. (Standard Methods, 18th edition)
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Limit of Quantitation (LOQ), or Lower Limit of Quantitation (LOQ), is the lowest concentration of the measurand that can be measured at a level of repeatability precision and trueness above which quantitative results may be obtained with a specified degree of confidence. The LOQ specifies the performance of a method and is mathematically defined as equal to 10 times the standard deviation of the results for a series of replicates used to determine a justifiable limit of detection. Limits of quantitation are matrix, method, and analyte specific. Moreover, any substance detected at a concentration greater than the LOQ is more than 99% likely to be present, and the quantitated value can be reported with a high degree of confidence so these substances are reported without qualification. (ss. NR 140.05(13) & 149.03(16)) Method Detection Limit (MDL), is the minimum concentration of a substance that can be measured and reported with 99% confidence that the analyte concentration is greater than zero, and is determined from analysis of a sample in a given matrix containing the analyte. Method detection limits are statistically determined values that define how easily measurements of a substance by a specific analytical procedure can be distinguished from measurements of a blank (background noise). MDLs are matrix, instrument and analyst specific which require a well-defined analytical method. In addition, these limits provide a useful mechanism for comparing different laboratories' capabilities with identical methods as well as different analytical methods within the same laboratory. Environmental Protection Agency's (EPA) MDL procedure1 promulgated at 40 CFR (Code of Federal Regulations) Part 136, Appendix B, rev. 1.11. The EPA defines the MDL as the "minimum concentration of substance that can be measured and reported with 99% confidence that the analyte concentration is greater than zero, and is determined from analysis of a sample in a given matrix containing the analyte". Understanding this definition is critical to understanding exactly what the MDL represents. Statistically, the 99% confidence interval means that any substance detected at a concentration equal to the MDL is 99% likely to be present at a concentration greater than zero. It also means that there is a 1% chance that a substance detected at the MDL will be considered (falsely) "present" when in reality the true analyte concentration is zero. This situation is known as a false positive, or Type I decision error, and the MDL procedure is designed to protect against making this type of error. The MDL is a statistical, rather than chemical, concept and it is quite possible that a substance can be "detected" at concentrations well below the MDL (hence the differentiation from the IDL). Also, the MDL tells us nothing about the numerical uncertainty of analytical results. It is assumed that because a substance was detected at a concentration equal to or greater than the MDL, that substance is 99% likely to be
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present and the quantitated value is the "best available estimate" of the true value. Quantitative uncertainty in a reported value exists to a greater or lesser extent in all analytical data, and must be accounted for with additional QC information. Because the data user does not necessarily know how an MDL was calculated, it is important to specify the proper units (mg/L or µg/L) and matrix type with all reported MDL data. Unfortunately, it is practically impossible to pinpoint a "true" DL for most analytes without introducing some uncertainty about the validity of low level results. It is important to note that the MDL is only a mechanism for dealing with analytical uncertainty. It is acceptable to round the calculated value up to the nearest decimal place. For example, if the calculated MDL is 0.15, it is acceptable to round the MDL to 0.20 if results are only reported to one significant figure. MDLs should never be rounded down, unless the laboratory feels it can routinely achieve the rounded value. (40 CFR part 136, Appendix B, rev. 1.11) Analytical Performance (AP), refers to how well an instrument or method can measure the analyte of interest – in other words, are results both reliable (reproducible) and valid (accurate) Quality Control (QC), is a procedure or set of procedures intended to ensure that a manufactured product or performed service adheres to a defined set of quality criteria or meets the requirements of the client or customer Quality Assurance (QA), is a way of preventing mistakes or defects in manufactured products and avoiding problems when delivering solutions or services to customers; which ISO 9000 defines as “part of quality management focused on providing confidence that quality requirements will be fulfilled”
2.
Why Detection Limits (DLs) are important for medical analyses? Medical laboratories are already and increasingly faced with the question of the possible presence of a
measurand for medical (tumor markers, infectious agents, carcinogens, pollutants) or legal reasons (drugs of abuse). More formal understanding of the LOD and LOQ is therefore needed and they are considered highly important parameters in the validation process. The proposed procedures in this study have proved useful for application in the field of clinical chemistry and promoted a standardized approach for estimating DLs of clinical assays.
3.
MDL vs. uncertainty Relative uncertainty is a good way to obtain a qualitative idea of the precision of data and results. All
measurements have a degree of uncertainty regardless of precision and accuracy. This is caused by two factors,
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the limitation of the measuring instrument (systematic error) and the skill of the experimenter making the measurements (random error). Although, MDL tells us nothing about the numerical uncertainty of analytical results but it is practically impossible to pinpoint a "true" detection limit for most analytes without introducing some uncertainty about the validity of low level results. Furthermore, it is important to note that the MDL is only a mechanism for dealing with analytical uncertainty. Obviously, uncertainty is introduced in all steps of the sampling, transport, storage and analysis of a sample. Any substance detected at a concentration greater than the MDL but less than the LOQ is 99% likely to be present, however the uncertainty in the quantitated value is unknown and the actual concentration is questionable. The determined concentration is reported to alert data users that the result is between the MDL and the LOQ. These numbers may be used with caution for compliance or regulatory calculations, but require additional substantiation.
4.
Chemicals and reagents The following items show the commercial sources and some characterizations of the purchased chemicals
that used in the method section: -
Absolute ethylene glycol (Ethylene glycol) (HOCH2CH2OH), analytical standard, ≥ 99.9% (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Acetylacetone (CH3)2CH2(CO)2 (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Ammonia aqueous (ammonium hydroxide solution) 28% NH3 in H2O, ≥ 99.99% (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Ammonium Oxalate ((NH4)2C2O4.H2O) salt, ACS reagent, ≥ 99% (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Butane standard commercial grade, 99% (Local source)
-
Calcium carbonate (CaCO3), ReagentPlus, 99% (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Calcium chloride (CaCl2·2H2O), ReagentPlus, ≥ 99.99% (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Calcium standard solution (1000 mg Ca2+/L = 25 mM Ca2+), Certipur, NIST Ca(NO₃)₂ in 500 mM HNO₃ (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Citric acid (HOC(COOH)(CH2COOH)2), ACS reagent, 100% (VWR, Radnor, PA, USA)
-
Detertec neutral (Vetec, Rio de Janeiro, Brazil)
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-
Doubly distilled deionized water (dd-DI H2O) (Milli-Q Ultrapure water specifications: 18.2 MΩ cm @ 25 °C, TOC ≤ 5 ppb, Bacteria ≤ 1 CFU/mL), which is non-absorbent under UV radiation, has been used throughout
-
Disodium ethylenediaminetetraacetic acid dehydrate (EDTA) (Na 2H2Y.2H2O), GR ACS salt, ≥ 99% (Merck-Millipore Co., Massachusetts, USA)
-
Ethylenediaminetetraacetic
acid
disodium
magnesium
tetrahydrate
salt
(EDTA-Na2Mg)
((NaOOCCH2)2NCH2CH2N(CH2COO)2Mg) was not purchased since EDTA-Na2Mg salt is unavailable and too expensive, therefore it was formulated in the lab -
EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid), BioUltra, ≥ 99% (SigmaAldrich Chemie Gmbh, Munich, Germany)
-
Eriochrome Black T (EBT), ACS reagent, indicator grade (Merck-Millipore Co., Massachusetts, USA)
-
Ethylene glycol monomethyl ether, (Methyl glycol) (Methy Cellosolve), EGME, (CH 3OCH2CH2OH), puriss. P.a., ACS reagent, reag Ph Eur grade, ≥ 99.5% (GC) (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Hydrochloric acid (HCl), BDH ARISTAR Ultra, 32-35% (VWR, Radnor, PA, USA).
-
Magnesium chloride (MgCl2.6H2O), ACS reagent, 99-102% (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Magnesium sulfate (MgSO4.7H2O), ACS reagent, ≥ 98% (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Methyl red, acid-base indicator grade (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Murexide (ammonium purpurate metal), ACS reagent, reag Ph Eur indicator grade (Merck-Millipore Co., Massachusetts, USA)
-
Nitric acid (HNO3) 65%, ISO grade, ≥ 99.5% (Merck-Millipore Co., Massachusetts, USA)
-
Oxalic acid ((COOH)2.2H2O), High Purity grade, 99.5-102.5% (GFS Chemicals Inc., Powell, Ohio, USA)
-
Oxide-free magnesium ribbon, High quality grade, 99.9% (Sherman Chemicals Ltd, Gillingham, UK)
-
Perchloric acid (HClO4) 60%, ACS reagent (GFS Chemicals Inc., Powell, Ohio, USA)
-
pH 4.0 and 7.0 buffer solutions (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Pluronic F-68 (Polyoxyethylene-polyoxypropylene) (C3H6O.C2H4O)X, (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Potassium chloride (KCl), ACS reagent, 99% (VWR, Radnor, PA, USA)
7
-
Potassium dihydrogen phosphate (KH2PO4), Suprapur, ≥ 99% (Merck-Millipore Co., Massachusetts, USA)
-
Potassium hydroxide anhydrous (KOH), ACS reagent, ≥ 85% and carbonate-free (Merck-Millipore Co., Massachusetts, USA)
-
Potassium permanganate (KMnO4), ACS reagent, 99% (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Potassium thiocyanate (KSCN), ACS reagent, ≥ 99% (Panreac, Barcelona, Spain)
-
Salmiac (ammonium chloride) (NH4Cl), ACS reagent, 99.99% (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Sodium bicarbonate (C6H4N3NaO8), ACS reagent, ≥ 99.7% (VWR, Radnor, PA, USA)
-
Sodium picrate monohydrate ((NO2)3C6H2ONa.H2O), SAJ first grade reagent, ≥ 95% (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Sodium chloride anhydrous (NaCl), ReagentPlus, ≥ 99% (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Sodium hydroxide (NaOH) anhydrous, ARG, ≥ 99% (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Sodium nitrite (NaNO2), ACS reagent, ≥ 98% (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Sodium sulfate decahydrate (Na2SO4.10H2O), ACS reagent, ≥ 99% (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Standard Reference Material (SRM-2670), simulated freeze dried urine control sample (National Institute of Standards and Technology (NIST), Gaithersburg, USA)
-
Standard Reference Material (SRM-2694), simulated rainwater control sample (National Institute of Standards and Technology (NIST), Gaithersburg, USA)
-
Strontium chloride hexahydrate (SrCl2), ACS reagent, 99% (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
20% Trichloroacetic acid (TCAA) (Cl3CCOOH), aqueous solution (VWR, Radnor, PA, USA)
-
Sulfuric acid (H2SO4), High Purity grade, 98% (PVS Chemicals Belgium NV, Ghent, Belgium)
-
TMP-SMX solution, 1:5 Trimethoprim:Sulfamethoxazole (100 mg/mL Dimethyl Sulfoxide (DMSO)), (Sigma-Aldrich Chemie Gmbh, Munich, Germany)
-
Urea (NH2CONH2), Ultra Pure grade, ≥ 99% (Merck-Millipore Co., Massachusetts, USA)
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5.
Materials Materials used in this study were listed as follows:
-
Desiccator nalgene, Thermo Scientific (Nalge Nunc Int., Rochester, NY, USA)
-
MF-Millipore membrane filter (0.45 µm, 47 mm) (Merck-Millipore Co., Massachusetts, USA)
-
Micromedic Model 2500 Automatic Pipette and Ependorf pipettes with disposable tips (Micromedics Systems, Division of Rohm and Haas, Horsham, PA, USA)
-
Reusable plastic beakers (Consolidated Plastics Inc., Stow, Ohio, USA)
-
Salivette tubes (Sarstedt AG & Co., Nümbrecht, Oberbergischer Kreis, Germany)
-
Thermo Scientific Nalgene low-density polyethylene (LDPE) bottles, (Cole-Parmer International, Vernon Hills, Illinois, USA)
-
Transparent microcentrifuge Eppendorf plastic tubes with attached cap lids (Jack Chen Biologix Plastics Co., Ltd., Shanghai, China)
6.
Apparatuses
-
Force 7 micro- centrifuge (Denver Instruments, Norfolk, UK)
-
Jenway PFP7 flame photometer (Jenway Gransmore Green Felsted, Essex, UK)
-
Microlyte 6 analyzer (Thermo Fisher Scientific Oy, Vantaa, Finland)
-
FAAS-novAA 400 P (Analytik Jena AG , Jena, Germany)
-
Panasonic Undercounter Laboratory Freezer (Panasonic Healthcare Corporation, Wood Dale, Illinois, USA)
-
Sartorius Cubis weighing balance (Sartorius AG, Gottingen, Germany)
-
Sentron pH system 1001 (Sentron Europe, Roden, The Netherlands)
-
Universal Oven UN30 (Memmert GmbH+Co. KG., Schwabach, Germany)
7.
Reagents preparation
-
Ammonium oxalate (0.1 M): 12.2 g (to 1 mg) of ammonium oxalate were dissolved by dd-DI H2O in 1000 mL volumetric flask
-
Ca2+ stock solution (Std
stock,Ca
2+
, 8 mg CaCO3/mL = 80 mM CaCO3 = 3.2 mg Ca2+/mL = 80 mM Ca2+): A
primary standard grade of anhydrous CaCO3 (FW=100.087) was dried in the oven at 105 °C for 90 min. 4 g (to 1 mg) of CaCO3 were accurately weighed and transferred to 500 mL volumetric flask. 10 mL of dd-DI
9
H2O were slowly added with 1:1 HCl solution through a funnel to the volumetric flask until effervescence ceased. That made all the CO32- dissolved and the resulting solution was completely cleared out. At this stage, the addition of HCl had converted the CaCO3 to CaCl2. 250 mL dd-DI H2O were added to the flask then this quantity boiled for few minutes to expel CO 2. The solution cooled and few drops of methyl red indicator were supplemented. The solution was adjusted to the intermediate orange color by adding few drops of 1:1 HCl. At last, the solution was quantitatively transferred and diluted to the mark of the volumetric flask with dd-DI H2O -
Mg2+ stock solution (Std
2+ stock,Mg ,
1.9 mg Mg2+/mL = 80 mM Mg2+): A primary standard solution was
prepared by dissolving 1.9 g oxide-free magnesium ribbon (to 1 mg) in 5 mL HCl (S.G.1.18, 6 M) then diluted to the mark of the 1000 mL volumetric flask with dd-DI H2O and mixed thoroughly -
Ca2+ ISA (ionic strength adjustor), 1 M KCl: dd-DI H2O was filled a 100 mL volumetric flask up to the halfway mark. 7.46 g (to 1 mg) of previously dried anhydrous KCl were added. The flask swirled gently to dissolve the solid, then the flask filled to the mark with dd-DI H2O, capped and upended several times to mix the solution
-
Mg2+ ISA, 4 M KCl: dd-DI H2O filled a 100 mL volumetric flask up to the halfway mark and 29.8 g (to 1 mg) of previously dried anhydrous KCl were added. The flask swirled gently to dissolve the solid then filled to the mark with dd-DI H2O, capped and upended several times to mix the solution
-
H2SO4 (2 M = 4 N): 11 mL of concentrated sulfuric acid were diluted up to 100 mL with dd-DI H2O
-
NaOH (1 M): 4 g (to 1 mg) of NaOH were dissolved in 100 mL of dd-DI H2O
-
NaOH (50 mM): 50 mL of NaOH (1 M) were diluted to 1000 mL with dd-DI H2O
-
NaOH, (1 mM): 1 mL of NaOH (1 M) was diluted to 1000 mL with dd-DI H2O
-
EDTA standard titrant (1 mM): 0.3723 g (to 1 mg) of EDTA were weighed then placed into a clean 1000 mL plastic beaker. The solid was dissolved in dd-DI H2O, transferred to a clean 1000 mL plastic volumetric flask then the solution diluted to the mark with dd-DI H2O. Since the obtained solution was unclear thus 2-3 drops of 50 mM NaOH solution were added and mixed thoroughly which were sufficient to clear the solution. After that, the solution was stored in PE bottle. Everyday upon titration, the titrant standard solution was standardized against Ca2+ certified standard solution 1000 mg Ca2+/L (25 mM Ca2+) and adjusted. So that, 25 mL of standard EDTA (1 mM) is equivalent to 1 mL of standard calcium solution (1000 mg Ca2+/L)
10
-
EDTA-Na2Mg solution: 1.179 g (to 1 mg) EDTA and 780 mg (to 1 mg) of MgSO 4.7H2O were dissolved in 50 mL dd-DI H2O
-
EGTA (2 mM): 722 mg (to 1 mg) EGTA weighted in a beaker and approx. 200 mL dd-DI H2O were added then the EGTA was suspended under stirring. After that, NaOH (10 M) was added until the precipitate has dissolved completely. After cooling down, the solution was transferred quantitatively to a 1000 mL volumetric flask with dd-DI H2O, filled up to the mark and mixed.
-
Eriochrome Black T sodium solution: 0.5 g (to 1 mg) of the dye were dissolved in 100 mL of EGME
-
HClO4 (50 mM): 5 mL of 60% HClO4 were diluted to 1000 mL with dd-DI H2O.
-
KMnO4 (25 mM): 3.95 g (to 1 mg) of KMnO4 were added to 250 mL of dd-DI H2O in a 1000 mL volumetric flask. The solution was stirred to dissolve then diluted to the mark with dd-DI H2O
-
KOH (1.25 M): 7 g (to 1 mg) of KOH were added to a volumetric flask containing 60 mL of dd-DI H2O. The solution was mixed to dissolve the content and cooled to the room temperature. After that, it was diluted to 100 mL with dd-DI H2O and mixed thoroughly
-
KOH (1.25 mM): 1 mL of KOH (1.25 M) was diluted to 1000 mL with dd-DI H2O and mixed thoroughly
-
Murexide indicator solution: 150 mg (to 1 mg) of the dye were dissolved in 100 g (to 1 mg) of absolute ethylene glycol
-
NH3-NH4Cl buffer solution (pH: 10.5) (8.5 M): 16.9 g (to 1 mg) of Salmiac were dissolved in 14.3 mL concentrated ammonia then diluted with 100 mL dd-DI H2O in a volumetric flask
-
Oxalic acid, 0.1 M: 12.6 g (to 1 mg) of oxalic acid were dissolved by dd-DI H2O in 1000 mL volumetric flask
-
Ammonium Oxalate/Oxalic acid mixture: 5 mL of oxalic acid (0.1 M) were added to 95 mL of ammonium oxalate (0.1 M) in a 100 mL volumetric flask
-
SrCl2, 0.0005% Sr: 0.0304 g (to 1 mg) of SrCl2.6H2O were dissolved in a volumetric flask filled up to 2000 ml with dd-DI H2O
-
Glassware rinsing solution: All glass components rinsed by soaking each of them in acidified (nitricpermanganate) solution so these components were treated at first by 50% HNO 3, washed by dd-DI H2O, followed by (25 mM) KMnO4 treatment; at last the glassware were rinsed several times again with dd-DI H2O
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8.
Ca2+ and Mg2+ elution from laboratory materials It is allowable for some analysts to neglect in practice a possible transfer of alkaline-earth ions from the
glass of the bottles to stock solutions of relatively high concentration, kept in them. However, when standard solutions are kept in these bottles for a longer period, an error may be made by neglecting this transfer completely. Therefore, it is necessary to investigate the quality of the bottle material used for the preservation of the standard solutions. In this sequence, we elaborated a method to investigate a possible transfer of Ca 2+ and Mg2+ from the glass of bottles to fluids kept in them. For this investigation, we used two different kinds of glass at our disposal, while polypropylene (PP) and PE bottles were also examined. The results of this investigation are shown in Fig. 1S; the correlation between the transfer and the time in days was indicated. Among these materials, PE has proved to be suitable to preserve the standard solutions required for our investigation for at least 1 year - the period of time covered by our research. The transfer of Ca 2+ and Mg2+, observed for PE was negligibly small, however, a correction factor should be considered when using glass ware.
Fig. 1S. The effect of laboratory collection material on Ca 2+ and Mg2+ increments in aqueous solution for a long span. Sample: Whole saliva, measurement by FAAS, n = 5, time of experiment: 360 days. Note: This test was carried out before launching the methods validations.
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9.
Chemical analysis of some biological fluids
Table 1S Biochemcial analysis of some biological fluids (n = 3). Components K+ (g/L)
Whole saliva (N = 10) 0.66-1.66
Periodontitis smoker saliva (N = 10) 0.27-0.36
Na+ (g/L)
0.06-0.23
0.19-0.21
SO42- (g/L)
2.2 ×10-34.7×10-3 0.19-2.11
2.0 ×10-33.8×10-3 0.12-1.40
1.0-2.8
4.3-5.0
1.40-1.92
1.12-1.55
PO43- (g/L) Total proteins (g/L) η (cP)
Plasma (N = 20)
CSF (N = 20) (1)
Sweat (N = 20)
Urine (N = 20)
Method
0.170.32 3.003.33 120-280
0.10-0.14
0.16-0.38
FAES
2.99-3.44
0.23-0.92
1.1 ×10-51.6×10-5 0.03-0.04
2.6 ×10-35.7×10-3 0.03-0.09
0.984.89 0.925.06 240-760
IC
0.1-0.5
0.3-0.9
2.563.88 0.2-1.6
0.090.12 0.4-1.0
FAES IC
Spec.
1.100.75-0.95 0.92-1.23 0.7-1.2 Ostwald 1.32 γ (mN/m) 45.2-50.6 40.8-46.1 43.568.4-73.6 71.4-75.7 55.0Capillary 50.2 68.3 rise TP: Total proteins, η: Viscosity, γ: Surface tension, Flame-Atomic Emission Spectroscopy: FAES, IC: Ion Chromatography, Spec.: Spectrophotometry Laboratory conditions: 25 °C and 55-65% RH (1)
Cerebrospinal fluid (CSF) is a clear, colorless body fluid found in the brain and spine. It is produced in the choroid plexuses of the ventricles of the brain. It acts as a cushion or buffer for the brain’s cortex, providing mechanical and immunological protection to the brain inside the skull. The CSF also serves a vital function in cerebral autoregulation of cerebral blood flow According to the outcomes of Table 1S, saliva was rich of K+, PO43-, proteins, with high viscosity and relatively low surface tension. Plasma was rich of proteins, Na+ and SO42- with high viscosity and low surface tension. CSF was just rich of Na+. Sweat had a relatively high viscosity and the highest surface tension. Urine was rich of Na+, K+, SO42-, PO43-, proteins and of high viscosity.
10. pH method validation To provide more reliability on pH measurements before sample analysis, it was decided to validate the pH method of analysis. Thus, a sensible scientific procedure based on the comparison between methods outcomes was adopted by measuring the pH of rainwater SRM-2694 sample by two different techniques. Hence, this standard sample was measured each time with ten replicates (n = 10) by a hand-held pH meter and by means of the micro-electrodes of Microlyte 6 analyzer. Soon after that, the mean value, standard deviation (SD), and relative standard deviation (RSD) were calculated for each procedure by the pH meter and by the microelectrodes of the analyzer. The RSD of each method, based on the replicate analysis of the QC (SRM-2694) material, was used to compare the reproducibility of the optimized methods.
13
11. pH method of analysis The pH values of the biological samples are dependent on the level of dissolved CO2. Hence, for a true pH value and to avoid any time-relating pH changes or loss of CO2, the degree of acidity was measured immediately after each sample collection, using a hand-held pH meter. Every day during the time of this research, the pH meter has been calibrated with reference buffers of pH 4.0 and 7.0.
12. EDTA standardization EDTA was standardized against Ca2+ certified standard solution (1000 mgCa2+/L) and adjusted each day before ions measurements. The true concentration of EDTA was assessed by using Eq. (1S): C EDTA, real =
𝐶𝑎 2+ 𝑐𝑒𝑟𝑡 ×𝑉 𝑆𝑎𝑚𝑝𝑙𝑒 𝑉 𝐸𝐷𝑇𝐴
(1S)
Where, [Ca2+cert] is the concentration (mM) of the certified standard solution of Ca 2+. VSample is the volume of the aliquot. VEDTA is the mean value (n = 5) of the consumed volume from EDTA (approximately 1 mM) solution.
14
13. Calibration series A series of standards was prepared for each analyte measurement by accurately pipetting the calculated volume of the corresponding standard stock solution (Std
stock,Ca
2+
or Std
2+ stock,Mg )
then diluting each aliquot to
2000 mL with dd-DI H2O in suitable volumetric flask (Tables 2S and 3S). Eight standard solutions were employed to create a calibration curve for each of the two ions and three of them were marked as low, medium, and high QCs. In specific, for each optimized method used for ion measurements, the correlation coefficient (R) of the relative calibration curve and its linear regression equation were defined. Table 2S Ca2+ calibration series. Sti Ca2+ (2)
St1
St2
St3
St4
St5
St6
St7
St8
V0,i (mL) (3)
5
12.5
27.5
37.5
52.5
65
77.5
97.5
Vfinal,Sti (mL) (4)
2000
2000
2000
2000
2000
2000
2000
2000
Sti conc. (mM)
0.2
0.5
1.1
1.5
2.1
2.6
3.1
3.9
Sti conc. (mg/L)
8
20
44
60
84
104
124
156
(2)
Eight standards of Ca2+ marked as: St1, St2, St3, St4, St5, St6, St7, and St8
(3)
V0,i (mL): Volume of Std stock,Ca2+ (3.2 mg Ca2+/mL = 80 mM Ca2+)
(4)
Vfinal,Sti (mL): Final volume of Ca2+ standard solution (Stdi Ca2+) after dilution
Table 3S Mg2+ calibration series. Stj Mg2+ (5)
St9
St10
St11
St12
St13
St14
St15
St16
V0,j (mL) (6)
2.5
5.0
7.5
12.5
15.0
17.5
22.5
25.0
Vfinal,Stj (mL) (7)
2000
2000
2000
2000
2000
2000
2000
2000
Stj conc. (mM)
0.1
0.2
0.3
0.5
0.6
0.7
0.9
1.0
Stj conc. (mg/L)
2.4
4.9
7.3
12.1
14.6
17.0
21.9
24.3
(5)
Eight standards of Mg2+ marked as: St9, St10, St11, St12, St13, St14, St15, and
St16 (6)
V0,j (mL): Volume of Std stock,Mg2+ (1.9 mg Mg2+/mL = 80 mM Mg2+)
(7)
Vfinal,Stj (mL): Final volume of Mg2+ standard solution (Stj Mg2+) after dilution
15
In similar way to the preparation method used to establish single calibration series for each cation, a combinational Ca2+ and Mg2+ calibration series had been created according to the specified application in Table 4S. This series was only used for Mg2+ measurements by EDTA titration. Table 4S Combinational Ca2+ and Mg2+ calibration series. Stc Ca2++Mg2+ (8)
St17(9)
St18(10)
St19(11)
St20(12)
St21(13)
St22(14)
St23(15)
St24(16)
V0,i (17)
2.5
12.5
27.5
37.5
52.5
32.5
77.5
97.5
V0,j (18)
2.5
7.5
7.5
12.5
12.5
7.5
17.5
17.5
Vfinal,Stc (mL) (19)
1000
2000
2000
2000
2000
1000
2000
2000
(8)
Eight combinational standards of Ca2+ and Mg2+: St17, St18, St19, St20, St21, St22, St23, and St24
(9)
St17 (8 mg Ca2+/L and 2.4 mg Mg2+/L)
(10)
St18 (20 mg Ca2+/L and 4.9 mg Mg2+/L)
(11)
St19 (44 mg Ca2+/L and 7.3 mg Mg2+/L)
(12)
St20 (60 mg Ca2+/L and 12.1 mg Mg2+/L)
(13)
St21 (84 mg Ca2+/L and 14.6 mg Mg2+/L)
(14)
St22 (104 mg Ca2+/L and 17.0 mg Mg2+/L)
(15)
St23 (124 mg Ca2+/L and 21.9 mg Mg2+/L)
(16)
St24 (156 mg Ca2+/L and 24.3 mg Mg2+/L)
(17)
V0,i (mL): Volume of Std stock,Ca2+ solution (3200 mg Ca2+/L)
(18)
V0,j (mL): Volume of Std stock,Mg2+ solution (984 mg Mg2+/L)
(19)
Vfinal,Stc (mL): Final volume of the combinational standard solution after dilution
14. Optimization of sample preparation method Sample preparation was accommodated to get rid of the interfering factors (i.e. K+, Na+, SO42-, PO43-, proteins, viscosity, and turbidity) that could weaken the response of the signal of the technique used for ion determination. The sample solutions were stored at -20 °C in 5 mL PP cryovials. After thawing, the solutions were only used during one measurement campaign and then discarded after 2 freezing/thawing cycles. Besides, to define the best method selectivity, the comparison of the LOQ and linearity between the optimized methods had been adopted.
16
15. Hand-held pH meter optimization The results of the pH values of the SRM-2694 sample measured by the pH meter and by the microelectrodes of the analyzer were expressed as mean ± SD (RSD). The measured values were in high agreement with the certified value where the outcomes obtained by the first technique achieved 3.5 ± 0.074 (2.1%) and by the second technique reached 3.5 ± 0.052 (1.5%). In addition, the confidence interval of the difference, t-value, df, and two-tailed P value were [-0.134,-0.026] at 95%, 3.1, 18, and 0.026, respectively. In comparison between these two criteria, the difference was considered to be very statistically significant. Therefore, the pH values of the samples were continually measured by the hand-held pH meter.
16. EDTA standardization The real EDTA concentration for 5 replicates found 1.152 ± 0.3 mM.
17. Calibration curves
Fig. 2S. Calcium calibration curve by EDTA titration.
Fig. 3S. Calcium and magnesium calibration curve by EDTA titration.
17
Fig. 4S. Calcium calibration curve by FAES technique.
Fig. 5S. Calcium calibration curve by ISE method: EMF vs. Log[Ca2+].
Fig. 6S. Magnesium calibration curve by ISE method: EMF vs. Log[Mg2+].
18
Table 5S Calcium and magnesium concentrations in biological fluids as found in the literature review. Cation Ca
2+
Mg2+
Sample
Concentration (mg/L)
References
Cerebrospinal fluid
132.3 ± 5.6
[3]
Periodontitis smoker saliva
43.2 ± 2.4
[4]
Whole saliva
86.8 ± 30.4
[5]
Sweat
44.6 ± 20.0
[6]
Plasma Urine Cerebrospinal fluid
93.2 ± 7.6 38.0 ± 0.72 27.5 ± 4.86
[7] [8] [9]
Periodontitis smoker saliva
5.83 ± 0.24
[4]
Whole saliva
30.9 ± 10.9
[5]
Sweat Plasma Urine
9.72 ± 4.86 10.2 ± 0.97 23.8 ± 7.78
[6] [10] [11]
Fig. 7S. Comparison of biological calcium contents between the outcomes of the current study (measurement by ISE method) and the literature review. Biological fluids are: (a) WS: Whole saliva, (b) PODSS: Periodontitis smoker saliva, (c) Pla: plasma, (d) CSF: Cerebrospinal fluid, (e) S: Sweat, and (f) U: Urine. Literature studies: Agha-Hosseini et al. [5], Zuabi et al. [4], Horecka et al. [7], Zuckermann and Glaser [3], Baker et al. [6], and Parentoni et al. [8]. The array of the 6 references meets the order of the 6 biological fluids.
19
Fig. 8S. Comparison of biological magnesium contents between the outcomes of the current study (measurement by ISE method) and the literature review. Biological fluids are: (g) WS: Whole saliva, (h) PODSS: Periodontitis smoker saliva, (i) Pla: plasma, (j) CSF: Cerebrospinal fluid, (k) S: Sweat, and (l) U: Urine. Literature studies: Agha-Hosseini et al. [5], Zuabi et al. [4], Mirčetić et al. [10], Liappis and Schneider [9], Baker et al. [6], and Arrabal-Polo et al. [11]. The array of the 6 references meets the order of the 6 biological fluids.
18. Difficulties
encountered
with
techniques
performances,
important
observations,
and
recommendations 18.1.
FAES
As we know, this technique is the most sensitive and convenient method of determining sodium (Na) and potassium (K) in different complex biomatrices as serum and urine, but it has not been broadly applied to the routine determination of alkaline-earth ions. At the same time, the nature of the biological samples which are rich in phosphates (PO43-) and proteins so vastly different that it offered an interesting and difficult testing ground technique. As a result of these complicated media with high viscosities and surface tensions, before FAES validation; the viscosity of the biological solutions had decreased the atomization rate considerably, which leaded to a weakening of the emission. The same influence was observed of the differences in temperature, which probably could also partly attribute to changes in viscosity. The composition of the coal-gas form of the municipal gas works was not always liable to great variations so that in uncontrolled conditions this gave a fluctuation in the temperature of the flame. However, using the background correction system (BCS) and after the optimization and the validation of FAES method and the sample preparation methods broadly predescribed in the current paper and through the uncertainty readings; the drift effects in FAES which may
20
influence the trueness and precision in a long series of measurements were found to have a negligible effect on measurements over time. This makes FAES a good micromethod for Ca2+ measurement with analytically sufficient repetitions over long periods. Despite the consequences of our findings, since a long time, due to the low sensitivity of FAES especially with the wide alterations when analyzing Ca 2+ in complex media as urine by this technique, there was a tendency to replace FAES by potentiometric procedures [12,13]. No wonder to say, that these teams where used uncontrolled procedures that made their analyses suffered from inferior accuracy and cumbersome separations of the interfering agents as higher concentrations of Na, K, sulfates (SO42-), PO43-, and proteins. We totally respect and accept their outcomes, but we say: there is no absolute candidate method; mainly our results have confirmed that there is no systematic difference between FAES and ISE methods which achieved a high r (0.9734) for Ca2+ measurements and proved this method under the optimized conditions that separated proteins and organic contents is tolerable in clinical chemistry. 18.2.
ISE
The advantages associated with the use of electrodes are the simplicity of the experimental setup, selectivity, sensitivity, fast response and relatively simple chemistry, which usually only comprise adjustment of ionic strength and pH. Nevertheless, the interpretation of the readout can be difficult if the ion to be determined exists in a partially complexed form in the sample solution, as the total content will then be quite different from the ionic activity sensed by the electrode. If the user of the ISE is skillful as she/he chooses a low timing of measurement (sampling rate: about 100 measurements/h) with the same sample volume at constant temperature through checking the reproducibility with a proper indication of the optimum choice of the degree of mixing, the readout can be very precise. Additionally, if the ISE user uses the flow injection analysis (FIA) mode so the electrodes in the flowing stream of carrier solution must be calibrated under exactly the same conditions (i.e., pumping rates, injected sample volumes, and composition of carrier electrolyte) as those used for actual analyses. Only under such conditions can the changes of the electrode potential be related via the respective ionic activities to the concentration of the analyte. So when using the FIA system, the composition of the carrier solution should be predefined to avoid any possible reaction between it and the sample. By this system, samples can be analyzed in a shorter span of time and all samples may be readily bracketed by sets of standards. On the other hand, the simultaneous measurement of Ca2+ and Mg2+ on one stream proved to give exactly the same results as separate duplicate measurements on identical samples.
21
18.2.1. ISE obstacles and avoidance We noticed when the ambient temperature is changed about 5 °C during a working day, corresponding to 1 mV in terms of the factor 2.3 RT/F, no problems are encountered. Fortunately, changes in laboratory temperatures are usually controlled. Therefore, in biological analyses strict precautions should be adopted and the composition of the carrier stream must be considered more carefully. However, the main two barriers of the analysis of the biological samples by repetitive measurements were indicated and avoided: (1) The possible adherence of the proteins to the electrode surface which affects the membrane potentials, so that is why we gave much interest to isolate proteins from the samples before ion measurement, and (2) The configurations of the electrodes were always maintained so that the more viscous samples were effectively washed away from the membrane surface. As a result of the optimized practices, we found that the slope of the calibration curves before and after analyses were unaltered and so was the potential constant (E0). By the way, for more complex biomatrices, the ISE user can stabilize the baseline and make the peak height value (PHV) analytically significant by spiking the electrolyte stream with the ions to be measured. Considering all the mentioned barriers and the emergent solutions, the validated ISE method and the preparation methods clearly specified in this paper; the responses of the electrodes for many biological samples analyzed in this study were not affected and these responses were linear over several decades of activity measurements which indicated that the electrodes surfaces were washed effectively. Consequently, even there are some users of these techniques do not suggest FAES as a candidate method for Ca2+ measurement in biological extracts, but we find the use of electrodes for this purpose has the obvious advantage of simplifying the instrumentation and decreasing the cost per analysis. References [1] N.W. Bower, Principals of instrumental analysis. 4th edition (D.A. Skoog: J.J. Leary), J. Chem. Educ. 69(8) (1992) 224. http://dx.doi.org/10.1021/ed069pA224.1. [2] A. Hubaux, G. Vos, Decision and detection limits for calibration curves, Anal. Chem. 42(8) (1970) 849–855. http://dx.doi.org/10.1021/ac6090a013. [3] E.C. Zuckermann, G.H. Glaser, Anticonvulsive action of increased calcium concentration in cerebrospinal fluid, Arch. Neurol. 29(4) (1973) 245–252. http://dx.doi.org/10.1001/archneur.1973.00490280057008.
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[4] O. Zuabi, E.E. Machtei, H. Ben-Aryeh, L. Ardekian, M. Peled, D. Laufer, The effect of smoking and periodontal treatment on Salivary composition in patients with established periodontitis, Periodontol 70(10) (1999) 1240–1246. http://dx.doi.org/10.1902/jop.1999.70.10.1240. [5] F. Agha-Hosseini, I.M. Dizgah, S. Amirkhani, The composition of unstimulated whole saliva of healthy dental students, Contemp. Dent. Pract. 7(2) (2006) 104–111. [6] L.B. Baker, J.R. Stofan, H.C. Lukaski, C.A. Horswill, Exercise induced trace mineral element concentration in regional versus whole-body wash-down sweat, Int. J. Sport Nutr. Exerc. Metab. 21(3) (2011) 233–239. [7] A. Horecka, A. Hordyjewska, T. Blicharski, J. Kocot, R. Zelazowska, A. Lewandowska, J. Kurzepa, Simvastatin effect on calcium and silicon plasma levels in postmenopausal women with osteoarthritis, Biol. Trace Elem. Res. 171(1) (2016) 1–5. http://dx.doi.org/10.1007/s12011-016-0635-1. [8] L.S. Parentoni, R.C.S. Pozeti, J.F. Figueiredo, E.C. de Faria, The determination of total calcium in urine: a comparison between the atomic absorption and the ortho-cresolphtalein complexone methods, Bras. Patol. Med. Lab. 37(4) (2001) 235–238. http://dx.doi.org/10.1590/s1676-24442001000400003. [9] N. Liappis, A. Schneider, Normal value of sodium, potassium, chloride, calcium, inorganic phosphate and magnesium in cerebrospinal fluid of children, Klin. Padiatr. 196(6) (1984) 370–374. [10] R.N. Mirčetić, S. Dodig, M. Raos, B. Petres, I. Čepelak, Magnesium concentration in plasma, leukocytes and urine of children with intermittent asthma,
Clin. Chim.
Acta
312(1-2) (2001) 197–203.
http://dx.doi.org/10.1016/S0009-8981(01)00622-2. [11] M.A. Arrabal-Polo, M.C. Cano-Garcia, G. Hidalgo-Agullo, L. Roletto-Salmo, Use of urinary concentrations in mg/dl in relation to absolute values in 24-hour samples for the evaluation of lithogenic factors in stone forming patients, Arch. Esp. Urol. 69(2) (2016) 53–58. [12] K.J.M. Rao, M.H. Pela-fin, S. Morgemtern, Advances in automated analysis, 1972 Technicon International Congress, v1, Mediid, 1973, pp. 33. [13] M. Vanko, J. Meola, Advances in automated analysis, 1372 Technicon International Congress, v1, Mediart, 1973, pp. 37.
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