D-erythro-Sphingosine-1-phosphate(S1P), a bioactive lysophospholipid, is an important mediator of inflammation, atherosclerosis, and cancer. S1P is proposed as a clinical biomarker and diagnostic variable in fundamental research, routine testing, and large-scale clinical trials due to its signaling transducer functions.

An automation friendly LC-MS/MS method with a calibration range of 10 to 400 ng/mL was developed and validated to support clinical trials using S1P as a biomarker.

Sample Extraction

Challenge in Quantitative Recovery

The bipolar structure of S1P presented a challenge during extraction due to its tendency to accumulate in the aqueous/organic interface.

Overall recovery was 91.3% for S1P and 109.5% for S1P-d7.

Extraction Method

• A 50-uL sample size was used with internal standard S1P-d7. 
• Recovery was improved by using a liquid-liquid extraction with 10% formic acid (FA) in methyl tert-butyl ether (MTBE) as a solvent. 
• Partial supernatant was dried, reconstituted, and injected into the LC-MS/MS system.

LC-MS/MS Method

LC-MS/MS Conditions

HPLC: Shimadzu LC-20AD

Column: Thermo Acclaim C8, 50x2.1mm, 5um

Mobile phase A: 0.1% formic acid in water

Mobile phase B: 0.1% formic acid in acetonitrile

Flow rate: 0.8 mL/min

MS/MS Detection

Mass Spectrometer: AB Sciex API 4000

Ionization mode: ESI positive ion mode

Source temperature: 500° C

Ion transition monitered:

S1P: 381 → 264

S1P-d7: 388 → 271

Results

S1P Calibration Curve

The assay showed a linear calibration range of 10 to 400 ng/mL. The curve was linear (R² > 0.998) using weighted 1/x² regression. 

Chromatography and Method Sensitivity

Representative chromatograms of the quality control at the lower limit of quantification (LLOQ-QC, 10 ng/mL) and blank surrogate matrix (2% bovine serum albumin [BSA] cleaned with active charcoal).

Matrix Effect

No matrix effect was observed when QCs prepared in matrix were compared with those prepared in neat solution

S1P Endogenous Concentration

The endogenous level of S1P in 6 lots of screened blank matrix ranged from 19.1 to 157 ng/mL. A single lot was quantified (150 ng/mL) and used to prepare MQC (endogenous level) and HQC (200 ng/mL + endogenous level) samples.

Conclusions

• A sensitive, selective, and automation friendly method capable of assaying S1P in human plasma was developed and validated to support clinical trials using S1P as a biomarker.
• Excellent recovery of S1P was achieved by adjusting the extraction solvent composition.
• The method could be applied to other phospholipids that pose similar challenges in quantitative recovery.

Acknowledgement

Guodong (Gordon) Gu, Michelle Black, Deping Cheng, Yinghe Li, Yifan Shi, Meng Fang, Lynn Maines
Alliance Pharma, Malvern, PA; Janssen Research and Development, Spring House, PA; Apogee Biotechnology Corporation, Hummelstown, PA

This study was financially supported by Apogee Biotechnology Corporation.

Alliance Pharma is pleased to announce that Zhiyang Zhao, PhD, has joined its leadership team as Chief Scientific Officer (CSO).

Dr. Zhao brings to Alliance Pharma over 20 years of pharmaceutical industry experience with special focus on metabolic and pharmacokinetic properties of small and large molecules in drug discovery and development. Dr. Zhao has previously held positions at Pfizer, GlaxoSmithKline, and Amgen. Most recently, Dr. Zhao served as Amgen’s Site Director for Preclinical Studies at Cambridge, Massachusetts, for over 11 years. He previously served as a Director at GlaxoSmithKline and as an Investigator at Pfizer. His extensive experience spans the entire drug discovery and development process (discovery, development, and in-/out-licensing) in oncology, metabolic, antiviral, CNS, and inflammatory diseases.

Dr. Zhao’s contributions and accomplishments of integrating in vitro and in vivo ADMET (absorption, distribution, metabolism, excretion, and toxicity) information to optimize drug candidate properties are documented in over 50 peer-reviewed scientific publications and patents in the areas of new drug targets, drug metabolism, pharmacokinetics, and toxicology. He currently also holds an appointment as Adjunct Professor at the Eshelman School of Pharmacy at the University of North Carolina at Chapel Hill, North Carolina, and serves as the Editor-In-Chief for Drug Metabolism Letters.

“We are thrilled to have Dr. Zhao on our scientific leadership team; he will further strengthen and broaden our scientific excellence in the services that we provide,” said Frank Li, PhD, Co founder and President of Alliance Pharma. “In his role as CSO, Dr. Zhao, will provide scientific, intellectual, and managerial leadership as well as explore scientific and business opportunities beyond Alliance Pharma’s current scope of business.”

Dr. Zhao expressed his excitement in joining the diverse team at Alliance Pharma, stating, “I look forward to working with the talented team at Alliance Pharma as we explore new business opportunities and expand our scientific and technical capabilities to better serve our clients. Having spent more than two decades in various leadership roles in pharmaceutical companies, I fully understand our clients’ challenges and needs.”

5-Hydroxymethylfurfural (HMF) is a water-soluble heterocyclic organic compound derived from sugars. HMF binds with high affinity to intracellular sickle hemoglobin (HbS). In vivo studies using transgenic sickle mice showed that orally administered HMF inhibits the formation of sickled cells in the blood. NIH and its collaborators conducted investigations into the possibility of HMF as a treatment of Sickle cell disease (SCD) which is characterized by an abnormality in the oxygen-carrying haemoglobin molecule in red blood cells. A method for the quantitation of HMF and one of its major metabolites, 5-hydroxymethyl-2-furoic acid (HMFA), has been developed and validated in commercially available human plasma by Alliance Pharma.

Method

The standards and QCs were spiked with stable-isotope labeled HMF/HMFA as internal standards and extracted by protein precipitation with 0.1% formic acid in acetonitrile in a phospholipid removal plate. The eluent was evaporated to dryness and the residue was reconstituted with acetonitrile:water (10:90). The analysis was conducted utilizing a Schimadzu Prominence 20AC HPLC system coupled with SRM detection in ESI positive mode for HMF and in ESI negative mode for HMFA on a Sciex API 4000 Q Trap mass spectrometer. Chromatographic separation was achieved using an Atlantis T3 column with 0.02% acetic acid in water and 4 mM ammonium formate in methanol as the mobile phases.

LC-MS Conditions

Chromatographic Conditions
HPLC: Shimadzu LC-20AC
Column:Waters, Atlantis T3, 100x2.1mm, 3 μm
Column Temperature: 40oC
Mobile phase A: 0.02% acetic acid in water
Mobile phase B: 4 mM ammonium Formate in methanol
Flow rate: 0.6 mL/min

MS/MS Detection

Mass spectrometer: Sciex API 4000 Q Trap
Source temperature: 550oC
Ion transition monitored:
HMF: ESI positive mode
m/z 126.9 → 53.1
HMFA: ESI negative mode
m/z 141.0 → 69.2

Precision and Accuracy of Spiked QCs for HMF

Spiked quality control sample precision and accuracy were demonstrated at n = 6 at the lower limit of quantification (5 ng/mL) in one validation run, and at low (15 ng/mL), medium (150 ng/mL) and high concentrations (1500 ng/mL) over three validation runs.

Precision and Accuracy of Spiked QCs for HMFA

Spiked quality control sample precision and accuracy were demonstrated at n = 6 at the lower limit of quantification (0.1 μg/mL) in one validation run, and at low (0.3 μg/mL), medium (6 μg/mL) and high concentrations (75 μg/mL) over three validation runs.

Conclusion

• A selective and sensitive HPLC-MS/MS method for the quantification of HMF and HMFA in commercially available human plasma was developed. 
• Great retention and selection of highly hydrophilic compounds were achieved using carefully selected HPLC column and optimized mobile phases.
• Phospholipid removal plate was used to decrease the ion suppression resulted from the phospholipids in the protein precipitation extract.
• The method was validated as linear, accurate, precise and reproducible.

Acknowledgements

Meng Fang, Yifan Shi, Yinghe Li, Michael Zhang, Bradley Gillespie, Warren Stern, Amy Wang, Nora Yang, and Xin Xu
Alliance Pharma, 17 Lee Boulevard, Malvern, PA 19355; Leidos Biomedical Research Inc., Frederick, MD 21701; AesRx, LLC, Newton, MA 02466; TRND, National Center for Advancing Translational Sciences, NIH, 9800 Medical Center Dr., Rockville, MD 20850

On Sunday, May 3, 2015, Alliance Pharma participated along with 40,000 people in Boston’s 47th annual “Walk for Hunger.” The 20-mile route wound around Boston and surrounding communities and raised money to create sustainable solutions for hunger in Massachusetts. This year’s walk raised more than $3 million. Alliance Pharma raised $500 and walked the whole 20 miles to support this great cause. Thank you for your donations and support!

In 2014, Alliance Pharma was recognized as one of the 100 fastest growing private companies in Philadelphia by the Philadelphia Business Journal. 

Rapid and sensitive measurement of cortisol and 6
-Hydroxycortisol in human urine using LC-MS/MS.

Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP metabolizing
enzyme. Cortisol can be converted to 6ß-hydroxycortisol (6ß-HC) by CYP 3A4. The latter is excreted in urine. Cortisol and 6ß-HC ratio in urine may act as CYP3A4 activity indicator.

Sample Preparation

• Human urine samples are spiked with internal standard and then extracted by liquid-liquid extraction with MTBE.
• Partial supernatant is dried, reconstituted, and injected into LC-MS/MS.
• STDs, LLOQ, and LQC are prepared in urine surrogate, PBS solution.

LC-MS/MS Method

Column: Aquity BEH C18, 50 X 2.1 mm, 1.7 μm
Mobile Phase: 0.1% formic acid in water and ACN
Flow Rate: 0.5 mL/min
Mass Spec: Sciex API 5500, ESI+

Results

Conclusion

A rapid and sensitive LC-MS/MS method was validated for cortisol and 6ß-HC analysis
in human urine. Method accuracy, precision, repeatability, selectivity, F/T stability, processed sample stability, bench-top stability, and long term stability have been confirmed.

Scientists

Guodong (Gordon) Gu, Yifan Shi, and Yinghe Li

The plasma concentration ratio of 4b-hydroxycholesterol (4b-HC) to cholesterol has been recognized as a reliable marker for the assessment of Cytochrome P450 (CYP) 3A4 activity. It could be a valuable yet simple and cost effective side-product of a clinical study to evaluate CYP3A4-mediated drug-drug interactions.

Detecting 4b-HC in biological matrices using LC-MS/MS has been a challenge due to its poor ionization efficiency and lack of a predominant daughter ion, which causes a low sensitivity. Isomeric metabolites of cholesterol and other sterollike endogenous interferences also pose additional challenges for LC-MS/MS method development. In this study, a sensitive and robust LC-MS/MS method was developed and validated for the simultaneous determination of 4b-HC and cholesterol in human plasma.

Sample Preparation

The plasma sample (50 μL) was hydrolyzed using potassium hydroxide prior to the liquid-liquid extraction (LLE) with hexane. The extract of LLE was incubated for 30-minute with picolinic acid, then extracted again using hexane. The final extract was split to two portions and analyzed using LC-MS/MS with separate methods. 4b-HC-d7 and cholesterol-d7 were employed as the internal standards.

LC-MS/MS Analysis

A nitrogen-containing moiety was introduced to 4b-HC and cholesterol molecules via a derivatization with picolinic acid. The enhanced ionization efficiency, and the formation of predominant product ions significantly increased the sensitivity of the SRM detection.

While the derivatization helped enhance the sensitivity, it significantly reduced the chromatographic baseline and eliminated the relevant interference peaks, which led to the successful chromatographic separation of 4b-HC within a 10-min run time. Five cholesterol metabolites, 4a-HC, 22-HC, 24-HC, 25-HC and 27-HC, were tested at 200 ng/mL for interference. None of these metabolites showed an interference peak at the retention time of 4b-HC, and the precision and accuracy of 4b-HC was not impacted.

Due to the high endogenous level of 4b-HC and cholesterol in blank plasma, a surrogate matrix (5% BSA) was used to prepare calibration standards. Precision and accuracy was evaluated by spiking known concentrations of analytes in the pre-quantified “blank” matrix.

The precision and accuracy of QC Samples were tested in three consecutive batches. The assay showed excellent linearity. The calibration curve was generated using a weighted 1/x2 regression. The endogenous concentrations of 4b-HC and cholesterol in more than 300 clinical samples were measured and revealed that 4b-HC ranged 12.5 -70.2 ng/mL, and cholesterol ranged 0.78 – 2.71 mg/mL.

Conclusions

• 4b-HC and cholesterol were quantified using only 50 μL of plasma samples in a semi-automated 96-
  well format.
• The method was validated as linear, accurate,
  precise and reproducible.
• The method has been successfully used to support several clinical studies

Scientists

Yinghe Li, Brock Fiorito, Jean Liu, Meng Fang, Yifan Shi and Michael Zhang

The purpose of this study was to develop and validate an LC-MS/MS method for assaying N-acetylmannosamine (ManNAc) in human plasma (K2EDTA) to support a phase I clinical study.

GNE myopathy, previously known as hereditary inclusion body myopathy (HIBM) is an autosomal recessive, muscular disorder, characterized by progressive muscle weakness with onset in early adulthood. ManNAc is being investigated by NIH and New Zealand Pharmaceuticals, as the precursor of sialic acid, prevents the development of muscle disease in the mouse model of GNE myopathy.

As an endogenous compound, calibration standards for analyzing ManNAc in human plasma need to be prepared in surrogate matrix. The presence of multiple hydroxyl groups makes it difficult to use reversed-phase chromatography due to the lack of retention. Alliance Pharma developed a HILIC LC-MS/MS method with a calibration range of 10 to 5000 ng/mL. The method has been validated thoroughly to support clinical trials of ManNAc in GNE myopathy patients. The research was funded by NCATS via NCI Contract No. HHSN261200800001E.

Method

ManNAc has four hydroxyl groups and relatively low molecular weight (221 amu). Its chromatography in various reverse phase columns had poor peak shape and low sensitivity. This problem was addressed by using an amide column in HILIC mode with trifluoroacetic acid (TFA) as the mobile phase modifier. The matrix background caused severe ion suppression which was probably due to the phospholipids in acetonitrile crashed human plasma. The suppression problem was solved by utilizing a phospholipid removal plate in the extraction step. Fifty microliters of human plasma was extracted with ManNAc-13C-d3 as the internal standard. No matrix effect was observed by comparing the results from QCs prepared in matrix and prepared in neat solution.

LC-MS Conditions

Chromatographic Conditions

HPLC: Shimadzu LC-20AD

Column: Waters XBridge Amide, 100 x 2.1 mm, 3.5 µm

Mobile phase A: 0.2% acetic acid & 0.05% TFA in water

Mobile phase B: 0.2% acetic acid & 0.05% TFA in acetonitrile

Flow rate: 0.8 mL/min

MS/MS Detection

Mass spectrometer: Sciex API 4000

Ionization mode: ESI positive

Source temperature: 400 °C

Ion transition monitored:

ManNAc: 222 → 126

ManNAc-13C-d3: 226 → 130

Results

ManNAc Calibration Curve  

The assay showed a linear calibration range of 10 to 5000 ng/mL. The curve was linear (R2 > 0.997) using weighted 1/x2.

Chromatogram and Sensitivity

Representative chromatograms of LLQC (10 ng/mL) and blank surrogate matrix (5% BSA). Retention time = 2.5 minute.

Precision and Accuracy of Spiked QCs

Spiked quality control sample precision and accuracy were demonstrated at n = 6 at the limit of detection (10 ng/mL) in one validation run, and at low (30 ng/mL), medium (251 ng/mL) and high concentrations (4051 ng/mL) over three validation runs.

Recovery

ManNAc was spiked in the surrogate matrix as well as in plasma at n = 6. The recovery was calculated as the ratio of the mean peak area to that of the post-spiked sample. The recovery in 5% BSA was found to be 92.4%, while the recoveries in the plasma (medium and high QCs) were 132% and 113%. The fact that ManNAc is an endogenous compound probably led to recoveries over 100% for plasma samples, since the post-spiked level could only be estimated based on the endogenous concentration in plasma.

ManNAc Endogenous Concentration 

Six lots of individual human plasma were quantified for the endogenous level of ManNAc. It ranged between 38.6 and 49.5 ng/mL. A large pool of plasma was assayed in six replicates (mean 50.5 ng/mL) and was used to prepared medium (200 ng/mL + endogenous level) and high (4000 ng/mL + endogenous level) QCs.

Conclusion

• A HILIC LC-MS/MS method capable of assaying ManNAc in human plasma was developed.
• As an endogenous compound, the calibration standards were prepared in surrogate matrix, while the QCs were prepared in surrogate matrix as well as human plasma.
• The method was demonstrated to be accurate, specific for ManNAc in human Plasma and reproducible.
• The assay has been successfully validated to support clinical trials of ManNAc in GNE myopathy patients.

Acknowledgements

Yifan Shi, Meng Fang, Michael Zhang, Yinghe Li, Amy Wang, Ed Kerns, Nuria Carrillo-Carrasco, Xin Xu, Selwyn Yorke, Bradley Gillespie

Alliance Pharma, Malvern, PA;  TRND, NCATS, NIH, Rockville, MD;  New Zealand Pharmaceuticals, Palmerston North, New Zealand

Leidos Biomedical Research Inc. (formerly SAIC-Frederick, Inc.), Frederick National Laboratory for Cancer Research, Frederick, MD

The purpose of this study was to develop and validate a rapid and sensitive LC-MS/MS method for measuring Goserelin in human plasma (K2EDTA).

Goserelin is a synthetic analogue of a naturally occurring luteinising-hormone releasing hormone (LHRH). With its ability to suppress production of sex hormones, Goserelin is particularly used in treatment of breast and prostate cancer. For identification and quantification of Goserelin in biological matrices, a method has been reported in rabbit plasma with a lower limit of quantification (LLOQ) of 0.1 ng/mL and an overall run time of 10 minutes. Due to the low level dosage of Goserelin, the challenge of analyzing and quantifying Goserelin at an even lower concentration has to be addressed. In this study, a rapid and sensitive HPLC-MS/MS method was developed and validated for the determination of Goserelin at an LLOQ of 40 pg/mL in human plasma.

Method

In order to remove the complex interferences in matrix and enrich the analyte of interest, Waters Oasis WCX µElution plate was used to extract Goserelin and internal standard from human plasma. The human plasma samples were spiked with Triptorelin as internal standard and extracted using solid phase extraction. The eluent was evaporated to dryness and the residue was reconstituted with acetonitrile:water:formic aicd (10:90:0.5). The analysis was conducted utilizing the Schimadzu Prominence 20AC HPLC system coupled with SRM detection in ESI positive mode on a Sciex API5500 mass spectrometer.  Chromatographic separation was achieved using a reverse phase column with 0.1% formic acid in water and 0.1% formic acid in acetonitrile as the mobile phases.  The peak of interest was well separated from interference peaks within a 4.0 minute run time.

Short-term Stability and Reproducibility

Short-term stability of Goserelin in human plasma was established for 4 freeze/thaw cycles at -70oC/room temperature and 25 hours at room temperature.  Reinjection reproducibility of the extracted samples was demonstrated by reinjecting standards and quality control samples stored at 6oC for 48 hours.

Conclusion

• A rapid and sensitive HPLC-MS/MS method for the quantification of Goserelin in human plasma was developed.
• Solid Phase Extraction was successfully used in order to remove the complex interferences in matrix and enrich the analyte of interest.
• The method was validated as linear, accurate, precise and reproducible. It can be used to determine the concentration of Goserelin in human plasma as low as 0.04 ng/mL using only 100 µL of sample.

Scientists

Meng Fang, Yinghe Li, Yifan Shi

Alliance Pharma has successfully completed testing and validation of the Meso Scale Discovery® (MSD) Human V-PLEX Aβ Peptide Panel 1 Assay (6E10).  With the completion of this validation, Alliance Pharma becomes the first global CRO to validate the Aβ V-PLEX Panel 1, and greatly expands its analytical capabilities in Alzheimer’s and Neurodegenerative Diseases.

The MSD Aβ V-PLEX Panel 1 (6E10) is a multiplexed sandwich immunoassay which measures three amyloid peptides of significance to Alzheimer’s disease: Aβ42, Aβ40, and Aβ38.  MSD neurodegenerative disease assays provide a sensitive method for measuring the levels of peptide and protein targets within a single, small-volume sample.  V-PLEX immunoassays are developed under rigorous design control and are fully validated according to “Fit for Purpose” principles.

Sensitive biomarker assays are proving crucial to the design and execution of clinical trials that target the earliest stages of Alzheimer’s disease.  The Aβ Peptide Panel 1 (6E10) kit is validated for human CSF and thus is ideally suited for this purpose.

“The addition of the Meso Scale Discovery® MSD Aβ V-PLEX immunoassay to our service portfolio will further help our sponsors in the development of drugs to treat neurodegenerative diseases” said Dr. Feng Li, President of Alliance Pharma.