Shimadzu Solutions for Application Notebook - Clinical Research - Application Notebook

Significance of topic

Modern analytical workflows in clinical research and forensic toxicology demand high sensitivity, specificity and throughput for monitoring pharmaceuticals, metabolites and toxic agents in complex biological matrices. Automated sample preparation and advanced LC-MS/MS or GC detectors minimize manual handling errors, accelerate turnaround and enable reliable quantitation at low concentration levels. Integrating online extraction, derivatization and multiplexed detection helps laboratories meet evolving requirements for therapeutic drug monitoring (TDM), endocrinology assays, doping control, toxicological screening and environmental exposure studies.

Study overview and objectives

This collection of application notes presents Shimadzu’s solutions and case studies covering: automated TDM workflows using CLAM-2000 coupled to LCMS-8040; evaluation of blood lysis methods for immunosuppressant assays; high-sensitivity psychoactive drug screening; rapid β-lactam antibiotic quantitation by LCMS-8060; automated metabolite profiling in plasma; quantitation of free urinary amino acids with creatinine normalization; online SPE-LCMS/MS assays of enzyme activities in dried blood spots; consolidated FIA/LCMS/MS of amino acids, acylcarnitines and steroids; high-throughput screening of illicit and polar doping agents; sub-minute cyanide detection by PESI; multi-target screening with MS/MS library matching; sensitive analysis of steroids and NSAIDs by LCMS-8050; and carbon monoxide determination in blood by GC-BID. Each note demonstrates method performance, instrument configurations and practical benefits.

Methodology and instrumentation

• Fully automated sample preparation with CLAM-2000 for protein crash, filtration, IS addition and direct injection into LCMS-8040/8060.
• Online SPE columns (e.g. POROS R1 trapping) coupled to triple-quadrupole MS for enzyme assays in blood spots.
• DPiMS-2020 probe electrospray ionization for direct screening of cyanide in serum without pretreatment.
• 2D-HILIC LC-MS/MS platforms for polar doping agent separation and detection with rapid polarity switching.
• High-throughput FIA/LCMS/MS with automated six-port switching for newborn screening analytes and steroids.
• GC-BID systems (Tracera + BID-2010) for CO measurement with oxidizing pretreatment and headspace injection.
• LCMS-8050 and LCMS-8060 triple quadrupole instruments with MRM and MRM-triggered MS/MS library search workflows.

Main results and discussion

• Therapeutic drug monitoring:
  
  • CLAM-2000 + LCMS-8040 achieved per-sample cycles as low as 9 min for antiepileptics and 7 min for antiarrhythmics.
  • Validated linearity (r²>0.995), accuracy ±15 %, precision ≤15 % across wide calibration ranges.
• Immunosuppressant assay:
  
  • Freeze–thaw at –80 °C and ammonium chloride lysis yielded >90 % recovery of tacrolimus, sirolimus and everolimus.
  • CLAM-2000 online pretreatment ensured reproducible quantitation of drugs bound to erythrocytes.
• High-sensitivity psychoactive drugs:
  
  • Simultaneous analysis of 60 psychotropic drugs in serum, whole blood and urine using CLAM-2000 + LCMS-8060.
  • Recoveries 70–110 %, linearity across four orders of magnitude and screening in <3 min per sample.
• β-lactam antibiotics:
  
  • MRM-based LCMS-8060 method quantitated five drugs in plasma with LOQs 0.7–6.0 ng/mL and 2 µL injection.
  • Protein-crash sample prep yielded satisfactory recoveries, precision ≤15 % and matrix effects manageable.
• Hydrophilic metabolites:
  
  • Fully automated MeOH precipitation via CLAM-2000 + LCMS-8040 quantified amino acids with IS normalization.
  • Results agreed with manual prep and external clinical lab data; RSDs <6 % for key biomarkers.
• Urine amino acids:
  
  • CLAM-2000 + LCMS-8040 measured 22 free amino acids and creatinine in urine; calibration r²>0.99 and LOQs 0.1–5 µM.
  • Total creatinine-normalized amino acid profiles of 28 volunteers revealed distinct metabolic groups.
• Enzyme activities in dried blood spots:
  
  • Online SPE LCMS-8050 measured lysosomal enzyme activities via selective fluorogenic substrates in <6 min.
• Integrated FIA/LCMS/MS:
  
  • Single-platform quantitation of amino acids, acylcarnitines and steroids from one DBS in 7 min with two injections.
• Forensic drug screening:
  
  • CLAM-2000 + LCMS-8040 assay quantified 18 illicit drugs with 14 isotope-labeled IS; linearity 20–200 ng/mL and P.E. 62–122 %.
  • 2D LC-MS/MS simultaneously analyzed highly polar doping agents including meldonium and adrenergic stimulants with LOQs <1 ng/mL.
  • PESI-DPiMS-2020 achieved 15 s cyanide screening in serum without pretreatment; in-source CID provided structural confirmation.
  • Multi-target screening with MRM plus library-based MS/MS confirmation delivered high confidence and quantitative accuracy.
• Steroid and NSAID analysis:
  
  • Shimadzu LCMS-8050 triple quadrupole provided LOQs 10–100 pg/mL for 14 steroids and NSAIDs with multiple reference ion ratios.
• Carbon monoxide:
  
  • GC-BID (Tracera + BID-2010) achieved sensitive headspace CO quantitation in blood; LOQs <2 ppm and CO-Hb (%) calculation.

Benefits and practical applications

• Automation reduces manual errors and improves reproducibility across complex assays.
• Online sample preparation modules and direct ionization techniques cut sample-to-analysis times and reagent usage.
• Broad analyte coverage from small drugs to polar metabolites and gases within unified platforms streamlines workflows.
• High sensitivity and low LOQs conserve precious sample volumes and enable early-stage biomarker detection.
• Library-based MS/MS confirmation and multiple reference ions bolster identification confidence in forensic and clinical settings.

Future trends and possibilities

Integrating AI-driven data processing and predictive maintenance will further elevate throughput and reduce downtime. Emerging ionization techniques, microflow LC-MS and advanced detector designs will push sensitivity limits. Online coupling with immuno- or enzymatic assays will expand multiplexed capabilities, while cloud-based instrument networks will facilitate remote monitoring, data sharing and regulatory compliance in global multi-center studies.

Conclusion

Shimadzu’s portfolio, spanning automated sample prep, flexible chromatography, high-performance triple-quadrupole mass spectrometers and innovative detectors, addresses diverse analytical challenges in clinical research, therapeutic monitoring and forensic toxicology. The demonstrated methods combine speed, sensitivity, accuracy and robustness, enabling laboratories to streamline workflows, enhance data quality and respond to evolving testing demands.

References

• US FDA, Guidance for Industry: Bioanalytical Method Validation (2001).
• Japan MHLW, Guideline on Bioanalytical Method Validation in Pharmaceutical Development (2013).
• Shimadzu Application Notes and Technical Manuals C10G-E055 series.
• WADA Technical Document: Harmonization of AAF Calculation for Carbon Monoxide (2017).