Author Introduction Carmen Dews The highly soluble protein, albumin, has many functions within the body. The pri- mary role of albumin is maintenance of blood plasma’s osmotic pressure although it also acts as a transport protein for drugs, metals, fatty acids, hormones and enzymes. Sodium is a major cation in extra-cellular fluids and participates in many physiological functions. Potassium is the primary intracellular cation found in the human body. The concentration of ions in intracellular and extra cellular fluids is maintained by an active transport mechanism. Zinc is essential for growth of body cells and the functioning of enzymes and exists at trace levels in blood plasma and albumin. Instrumentation Flame Control • Fully programmable flame controller with safety interlocks All sample analyses were performed using the Agilent 240 FS double-beam AA spectrometer, which has the following • Fuel and oxidant gas flows set from PC specifications: • Gas control provides instantaneous gas flow change for fast sequential operation Optics • Automatic changeover between air and nitrous oxide • All reflective optics for optimum transmission efficiency flames as well as automatic recall and setting of optimum • Fully sealed double-beam optics with hard quartz over- gas flows for each element. coated mirrors • Separate flame-on and flame-off buttons • A single beam splitter with a rotating beam combining • Eight safety interlocks and separate upper and lower mirror for maximum light transmission and low noise flame shields Monochromator Spraychamber • Czerny-Turner 0.25 m focal length monochromator with • External manual adjustment of impact bead (without microstepping driver for enhanced resolution tools), for complete control over sensitivity and • Holographic grating with 1200 lines/mm atomization interferences • Wavelength range 185-900 nm • PTFE impact bead available for applications where • Computer controlled wavelength and slit selection with hydrofluoric acid is used automatic peaking on each analytical wavelength • Impact bead is available for use with both air-acetylene • Wavelength repeatability ± 0.04 nm or better and nitrous oxide-acetylene flames • Fluorinated high density polyethylene construction Lamp Control and Power Supplies capable of handling acids, alkalis and organic solvents • The power supply runs in two modes. The first mode is a two-lamp power supply; one for the operating lamp and • Fitted with an adjustable nebulizer with inert another for the automatic warm-up of the next lamp in platinum/iridium capillary and PEEK venture for corrosion sequence. The second mode powers four lamps simultane- resistance ously, enabling fast, sequential, multi-element per sample Burner operation • Designed for high solids operation with minimal carbon • User selectable option to leave lamps on or automatic build-up and burner blockage switch off at the end of an auto run • Universal design suitable for both nitrous oxide-acetylene • Lamps are electronically modulated for better sensitivity and air-acetylene and extended lifetime • PTFE corrosion shield • Four fixed lamp positions with fast automatic lamp selec- tion (conventional lamp turret is slow) to enable fast Atomizer Mounting and Adjustment sequential multi-element determinations sample by sample • Atomizer mounting mechanism suitable for flame, vapor and furnace AAS with corrosion resistant construction • Lamps fit directly into the lamp sockets without the need for corrosion prone restraining clamps or leads and • Burner rotation through a full 90 degrees without having electrical connectors to remove flame shields or extinguish the flame Background Correction • High speed deuterium background corrector for accurate correction of background signals • Correction range 185 to 425 nm to a total 2.5 absorbance • Electronic modulation with automatic gain attenuation for improved beam balance
2 Materials and Reagents Conditions Trace metal grade acetic acid (trace metal acetic acid, The analysis conditions used in this work are described in glacial (99.4%), Fisher Scientific, USA) and deionized water Table 3. (18.2 MW Barnstead, Dubuque, IA, USA) were used for the Table 3. AA Analysis Conditions sample and standards preparations. The standards were pre- pared by diluting multi-element stock standards (K, Na, and Na K Zn Zn, 1000mg/L, Inorganic Ventures, Lakewood, NJ USA). A Wavelength (nm) 589.6 766.5 213.9 stock QC (6020, 20mg/L, Inorganic Ventures, Lakewood, NJ, Gas used Air/Acetylene Air/Acetylene Air/Acetylene USA) was used for calibration verification. All samples, cali- Bandpass (nm) 0.2 1 1 bration standards, and QC’s were diluted with 5% v/v acetic acid. Fuel flow (L/min) 2 2 2 Mode Emission Emission Absorption D2 correction No No Yes Sample Preparation Replicates 3 3 3 Calibration and QC solutions were prepared as summarized in Table 1. The albumin samples and dilution factors are detailed Results and Discussion in Table 2. Table 4 details the original estimates of sample concentration Table 1. Calibration and QC Solutions provided by the customer as well as the sample concentration results obtained with the Agilent 240 FS AA spectrometer. Standard Name Element Concentration (mg/L) Blank K, Na, Zn 0 Table 4. Customer-Provided Estimates and Agilent 240 FS Spectrometer Sample Results Standard 1 K, Na, Zn 0.5 Standard 2 K, Na, Zn 1.0 Customer-provided 240 FS QC1 Na, K 0.5 concentration concentration Sample id Element (mg/L) (mg/L) %RSD QC2 Zn 0.2 LA 05D Na 2500–3500 3090 0.3 LA 05D K 3–5 4.15 0.5 Table 2. Sample Preparations LH Na 3000–4000 3570 0.1 LH Zn 0.2–0.5 0.267 0.8 Sample id Element Dilution AHF Na 2500–3500 3080 0.1 LA 05D Na 5000x LA 05D K 10x LH Na 5000x Several samples were spiked to verify the instrument perfor- LH Zn 2x mance in the albumin matrix. Sample LA 05D was spiked at 0.3 mg/L for Na. Sample AHF was spiked at 0.2 mg/L for Na. AHF Na 5000x Sample LH was spiked at 0.5 mg/L for Zn. The spike recovery results are listed below in Table 5. Duplicates of sample LH were analyzed for Na and sample LA 05D for analyzed for K as The samples listed were 25% albumin and were submitted by a further indication of the stability of the Agilent 240FS. a customer for analysis on the Agilent 240 FS. The sample dilutions used in this work were based upon the customer- provided estimates of the concentration of K, Na, and Zn.
3 Table 5. Spike and Duplicate Results (on Diluted Sample)
Sample id Na (mg/L) K (mg/L) Zn (mg/L)
LA 05D 5000x Na 0.619 LA 05D 5000x Na 0.3 ppm spk 0.939 Spike recovery % 106.7 LA 05D 10x K 0.415 LA 05D 10x K dup 0.409 LH 5000x Na 0.715 LH 5000x Na dup 0.719 LH 2x Zn 0.134 LH 2x Zn 0.5ppm spk 0.635 Spike recovery % 100.3 AHF 5000x Na 0.615 AHF 5000x Na 0.2 ppm spk 0.828 Spike recovery % 106.4 The QC samples were analyzed after the calibration and at the Curve fit linear origin end of the run. The results for the QC samples are as follows. Characteristic conc: 0.0050 mg/L Table 6. QC Results (mg/L) Correlation coefficient 0.9984
Sample id Na K Zn QC sample after calibration 0.509 0.503 0.199 % Recovery 101.8 100.5 99.4 QC sample at the end of run 0.517 0.51 0.201 % Recovery 103.4 100.8 100.6
The calibration curves are detailed below; the curves are
4 References L. H. J. Lajunen, and P. Peramaki, Spectrochemical Analysis by Atomic Absorption and Emission. Royal Society of Chemistry; Cambridge; 2004.
US Department of Health and Human Services, National
Institutes of Health website. Available at: www.nih.gov. Accessed 10th July 2006.
Method detection limits were determined by analyzing seven
replicates of the low standard (0.5 mg/L) and multiplying the standard deviation of the seven replicates by three. The method detection limits are listed in Table 7. Table 7. Method Detection Limits
Element Method Detection Limit (mg/L)
Na 0.008 K 0.009 Zn 0.001
Conclusion The results obtained illustrate that the Agilent 240 FS AA system excels at providing a simple and effective solution for the determination of K, Na, and Zn in albumin. The method has shown to be accurate and repeatable.
The QC samples are within ± 10% of the true value. The spike recoveries and duplicate samples show recoveries from 90–110%. The duplicates samples are within 5% of each other.
The cookbook settings provided in the software were used
for the analysis of Na, K, and Zn. All of the sample results fall within the expected ranges.
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