Determination of Calcium Magnesium in Blood Serum by Automated Flame Microsampling Application Note
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1. e o e e Author Lucinda M Voth Determination of Calcium and Magnesium in Blood Serum by Automated Flame Microsampling Application Note Atomic Absorption Introduction The number of trace metallic elements of biological importance is increasing and a wide range of elements need to be analyzed in blood urine and tissues There is often a natural limitation of sample volume particularly in the analysis of blood and tissue digest samples For example serum calcium measurements may routinely be required on sample volumes of 0 05 0 10 mL Therefore a means of minimizing sample consumption while retaining high sensitivity for the measurement is of con siderable importance For elements such as calcium and magnesium flame microsampling provides the necessary sensitivity and very small sample volumes can be analyzed with minimal sample preparation A further advantage of flame microsampling is that it minimizes the problems associated with the aspiration of high protein content solutions encountered in serum analysis j Agilent Technologies Determination of Calcium and Magnesium in Blood Serum Unassayed normal serum samples were analyzed for calcium and magnesium A serum volume of 100 uL was diluted to 5 mL and potassium was added to give a final concentration of 5000 pg mL in order to suppress ionization The sample was acidified with con centrated HCI The nitrous oxide acetylene flame was used for both analyses to over2. Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 1981 Printed in the USA November 1 2010 AA015 gi Agilent Technologies
3. measurements of the solutions ranged from 1 1 to 2 1 RSD The first peak in each set of six is the sample flush it is disregarded in the calibration and sample measurements This sample flush and 10 sec distilled water rinse between standards and samples eliminates the effects of carry over ZERO Cc 0 000 1 5 STD 1 C s00 STD 2 c 10 00 STD3 c 15 00 01 c 3 86 02 G 9 62 03 C 9 68 04 Cc 9 45 05 c 941 i MEAN 9 60 il 01 C814 02 G 8 06 03 G 8 02 I 04 C 834 i 05 C 8 24 MEAN C 8 16 The magnesium determination was carried out at the 285 2 nm line without background correction The PSC 55 parameters were the same as those used for the calcium determinations however only one sample was analyzed Calibration was again established directly in mg dL The actual standard concentrations were 0 2 0 4 and 0 6 pg mL magnesium As Figure 2 indicates the precision was excel lent from 1 1 1 4 RSD The determined concentration of 1 90 mg dL magnesium is within the normally expected serum magnesium range of 1 5 5 8 mg dL Routine Determination of Serum Calcium and Magnesium It has been shown that flame microsampling is a feasible method of measuring serum calcium and magnesium However long term accuracy and precision must be consid ered in the routine analyses of larger numbers of samples An assayed control serum was used to determine the long term accuracy and precision The standard concentrati
4. with the PSC 55 the operator can program a drift monitoring or calibration reslope after any selected number of samples 1 1 RSD ZERO C 0 000 STD 1 C 1 00 1 4 12 STD 2 C 200 m STD3 C 3 00 01 C 1 91 1 o 02 191 4 Tee 03 C191 04 C 187 ODA 05 C 488 MEAN C 1 90 Figure 2 Magnesium in blood serum 3 sai E TERETE i 1 ye aT WaN VEHET in i ii ul Aa eh Ai EN HH At il NU Hla Ki th W i NAGA AUAUAUAUANIU GN Conc 1 0 2 0 0 01 2 01 mg dL BIK Standard 2 Figure 3 Serum magnesium 0 2 0 4 0 6 pg mL Mg standards 1 0 2 0 and 3 0 mg dL serum mg 3 Table 1 summarizes the results of the magnesium determina tion The normal control assayed value was 2 4 mg dL with an expected range of 2 standard deviations 0 4 mg dL The mean value obtained was 2 15 mg dL The highest individual value was 2 21 mg dL and the lowest value was 2 10 mg dL Standard calibration and the 30 determinations took 30 min utes The analysis was repeated in a shorter period of time 15 min with a distilled water rinse after every 10 samples rather than after every sample with only a slight decrease in precision Table 1 also shows the results of the calcium determination The normal control assayed value was 10 0 mg dL with an expected range of 0 4 mg dL The highest individual value was 10 1 mg dL and the lowest value was 9 6 mg dL Standard calibration and the 30 determinations were com plete
5. come chemical interferences The dipping method of flame microsampling utilizing the Agilent PSC 55 Programmable Sample Changer was used The PSC 55 was programmed with the following parameters Number of standards 3 Number of samples 2 Rinse rate 1 A distilled water rinse after every sample Rinse time 10 sec Dip time 0 2 sec Multiples 5 Reslope rate 0 Reslope function was not used Sample flush 1 Sample flush preceding every standard and sample Calcium in Blood Serum STD3 1 1 RSD STD 2 2 1 STD 1 3 BLK Figure 1 Calcium in blood serum An Agilent AA 875 Atomic Absorption Spectrophotometer equipped with an adjustable uptake nebulizer was used The uptake rate was adjusted to 7 mL minute Integration time was 8 sec Measurements were made in the peak height mode The calcium determination was carried out at the 422 7 nm line Background correction was not necessary Calcium stan dards of 1 0 2 0 and 3 0 pg mL were prepared Standards also contained 5000 g mL potassium and were acidified with HCI Calibration was established taking into account the dilu tion factor to give results directly in milligrams per decilitre mg dL Figure 1 shows calibration of 3 standards and the calculated mean values for the two samples Measurements of 9 60 mg dL and 8 16 mg dL Ca are within the normal serum calcium concentration range of 7 5 to 13 0 mg dL The precisions for 5
6. d in 30 minutes with a distilled water rinse after every sample Table 1 Normal control No of Element assayed value samples Magnesium rinse after every sample 2 4 0 4 mg dL 30 rinse after 10 samples 2 4 0 4 mg dL 30 Calcium rinse after every sample 10 0 0 4 mg dL 30 Conclusion Flame microsampling utilizing the Agilent PSC 55 Programmable Sample Changer is an excellent method for determining calcium and magnesium concentrations in small volumes of blood serum The results were accurate and showed excellent precisions during the analysis of 30 samples The same solutions could be used for the AA determination of the electrolytes sodium and potassium if cesium rather than potassium is used for ionization suppres sion Flame microsampling minimizes sample preparation permits the determination of calcium and magnesium on serum samples of 0 10 mL and minimizes any clogging effects encountered in continual aspiration of high protein concentrations in serum Highest value Mean value lowest value Time 2 21 2 15 mg dL 2 10 30 min 1 4 RSD 2 15 mg dL 2 10 15 min 10 1 9 8 mg dL 9 6 30 min 1 5 RSD For More Information For more information on our products and services visit our Web site at www agilent com chem www agilent com chem Agilent shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material
7. ons and the sample dilution factor were the same as the previous determinations One serum sample dilution was carried out and analyzed 30 times so that inaccuracies could be attrib uted to the AA method of analysis rather than to dilution errors The following PSC 55 parameters were used Number of standards 3 Number of samples 30 Rinse rate 1 Rinse time 10 sec Dip time 0 2 sec Multiples 2 Reslope rate 10 Sample flush 1 The spectrophotometer parameters were the same as those used for the previous determinations of calcium and magne sium except that the integration time was reduced to 6 sec Figure 3 shows the recorder tracing for the magnesium cali bration and the first 20 samples The first peak of each stan dard and sample is the sample flush The calibration and sample measurements were determined from the mean of two readings After the first 10 samples the blank and second standard were rechecked This was done by selecting 10 as the reslope rate parameter on the PSC 55 The sample probe returns to the blank solution and then to a reservoir of the standard selected for reslope The actual reslope is achieved by the HP 85 computer accessory In this analysis drift was not significant enough to require res lope This programmed remeasurement of the blank and second standard can be used as a monitor of drift of both baseline and sensitivity Depending on the amount of drift encountered and number of samples up to 67
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Determination of Calcium Magnesium in Blood Serum by Automated Flame Microsampling Application Note