Determination of Calcium in Serum Samples by AAS Using a Fuel Lean Flame

Importance of the Topic

Accurate quantification of serum calcium plays a critical role in clinical diagnostics, informing patient assessment in endocrinology, nephrology and bone metabolism. Flame atomic absorption spectroscopy (AAS) offers the specificity and sensitivity required to distinguish free and complexed forms of calcium in serum, but matrix interferences demand careful optimization of flame conditions and sample preparation.

Objectives and Overview of the Study

The study aimed to develop and validate a flame AAS method that consistently recovers certified serum calcium standards (NIST SRM 909 a1, a2) within published ranges and maintains a maximum allowable error of less than 2%. A second goal was to compare air–acetylene lean flames with fuel-rich and nitrous oxide–acetylene flames in terms of accuracy, precision, sensitivity and detection limits.

Methodology and Instrumentation

Instrumentation and Cleaning Protocols:

• Agilent SpectrAA-20 Plus atomic absorption spectrometer with Mark VI burner
• Hi-Vac nebulizer set to high vacuum and twin mixing paddles for improved precision
• Photron HC hollow cathode Ca lamp (P809), lamp current 3 mA, wavelength 422.7 nm, slit width 0.5 nm
• Daily sonication of burner, spray chamber and nebulizer in deionized water with 0.01% Triton X-100 and acid washing of glassware

Sample and Reagent Preparation:

• Stock calcium solution from SRM 915 calcium carbonate; working standards spanning 0–14 mg/dL (0–2.8 µg/mL in 50× dilution)
• Lanthanum oxide releasing agent (1 g/L La in 0.25% HCl) to dissociate protein-bound calcium and suppress phosphate interference
• Sodium and potassium chlorides added to standards and diluent to mimic serum ionic strength
• Serum and standards diluted 50× in lanthanum diluent; bracketing with lower and upper standards and calculation by standard slope equation

Optimized Flame Conditions:

• Air–acetylene lean-blue flame: air flow ~14 L/min, acetylene ~2 L/min
• Burner height: 5–6 mm, aspiration rate: 5 mL/min, background correction off, delay time 5 s, measurement time 3 s, four replicates per sample

Main Results and Discussion

Comparison of Flame Conditions:

• Fuel-rich air–acetylene flames under-recovered SRM 909 a1 and a2 by several percent, while aqueous SRM 3109 remained accurate
• Lean air–acetylene flames yielded grand mean values of 9.31 mg/dL and 13.39 mg/dL for SRM 909 a1 and a2, matching NIST certified values with total CV <1%
• Nitrous oxide–acetylene flames improved raw sensitivity by ~2× but exhibited 5× worse precision due to high analyte emission noise

Linearity and Detection:

• Method linear between 0 and 2.8 µg/mL (50× diluted)—r = 0.9999, slope 0.0188, negligible intercept
• Optimal detection limit achieved with lean air–acetylene flame, balancing sensitivity and precision

Benefits and Practical Applications

The optimized lean-flame AAS method provides:

• High accuracy and precision for routine serum calcium measurement in clinical and quality-control laboratories
• Robust matrix tolerance through lanthanum release chemistry and ionic strength matching
• Rapid sample throughput with minimal interference correction

Future Trends and Applications

Emerging opportunities include:

• Adoption of high-temperature flames or alternative atomization sources (microwave-induced plasma, graphite furnaces) to further reduce interferences
• Integration of diode-laser AAS and wavelength modulation to improve signal-to-noise ratios
• Coupling with flow injection and chemometric algorithms for automated matrix correction
• Potential expansion to multi‐element clinical panels on compact AAS platforms

Conclusion

A lean air–acetylene flame in flame AAS, combined with lanthanum releasing agent and ionic strength modifiers, delivers accurate, precise and linear measurement of serum calcium. This method outperforms fuel-rich and nitrous oxide–acetylene flames in clinical serum matrices, meeting stringent error criteria and supporting high-throughput laboratory workflows.

Reference

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