TITLE: BC derivatization for dissolved marine metabolites 
AUTHORS: Brittany Widner, Melissa Soule, Frank Ferrer-Gonzalez, Mary Ann Moran, Elizabeth Kujawinski

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Benzoyl chloride derivatization of dissolved metabolites in seawater and culture medium improves sensitivity, number of quantifiable compounds, and sample throughput. Samples are quantified using a standard curve and stable isotope labeled internal internal standards prepared from an isotopically labeled reagent. Samples are filtered (0.2 um), derivatized in basic solution and extracted from seawater/culture medium using a solid phase extraction resin (PPL, Bond Elut, Agilent). Benzoylated metabolites are detected using UPLC-MSMS with Orbitrap detection. Peaks are analyzed with Skyline.</div><div class = "text-block">This version corrects typographical errors and a bug at protocols.io that caused videos to be lost.</div></div>

[STEPS]
?. [Prepare Standards for 1 mL and 25 mL methods]
Standards for samples (culture samples)
1 mL
?. [Prepare Standards for 1 mL and 25 mL methods]
1ostocks: mixes 1, 2, 3, 4, 5-6, and thiol* (optional).
?. [Prepare Standards for 1 mL and 25 mL methods]
2ostock:mix of all metabolites. 1:100 dilution of primary into matrix.
?. [Prepare Standards for 1 mL and 25 mL methods]
3ostock:  mix of all metabolites. 1:200 dilution of secondary into matrix.
?. [Prepare Standards for 1 mL and 25 mL methods]
Select concentration for Stable Isotope Labeled Internal Standards (SIL-ISs). Concentrations (and sensitivities) range over orders of magnitude, and you want to add a spike of the SIL-ISs that is detectable but not crazy high for every compound. The options are as follows.A) Make a standard mix with different concentrations for different metabolites based on their LODs. This is very cumbersome.B) Run each sample twice – once with a spike of  and once with . This necessitates the preparation of two standard curves: one from  to  (with   spike) and one from  to   (with  spike). C) Pre-run the samples (without adding the SIL-ISs) to estimate concentrations and then prepare standard curve and SIL-IS addition appropriately. This will still likely result in the need for either A or B, so I do not recommend this step.Note: Samples that exceed  will require dilution and reanalysis. Calibration curves for many metabolites are not linear above this concentration.
?. [Prepare Standards for 1 mL and 25 mL methods]
Prepare standards and MQ blanks in duplicate. Due to matrix effects, the MQ blank cannot be used quantitatively as a 'blank' but rather is used to identify contamination (e.g. in the reagents or vials) in a particular sample batch.
?. [Prepare Standards for 1 mL and 25 mL methods]
Volume additions for standards.  ABCD1  [Standard] (pg/ml)
  Volume matrix (uL)Vol 2o stock (uL)  Vol 3o stock (uL)
  2  0
    1000
    -
    0
  3  5
    990
    -
    10
  4  10
    980
    -
    20
  5  35
    930
    -
    70
  6  70
    860
    -
    140
  7  100
    800
    -
    200
  8  350
    300
    -
    700
  9  700
    993
    7
    -
  10  1000
    990
    10
    -
  11  3500
    965
    35
    -
  12  7000
    930
    70
    -
  13  10000
    900
    100
    -
  14  30000
    700
    300
    -
ABCD1  [Standard] (pg/ml)
  Volume matrix (uL)Vol 2o stock (uL)  Vol 3o stock (uL)
  2  0
    1000
    -
    0
  3  5
    990
    -
    10
  4  10
    980
    -
    20
  5  35
    930
    -
    70
  6  70
    860
    -
    140
  7  100
    800
    -
    200
  8  350
    300
    -
    700
  9  700
    993
    7
    -
  10  1000
    990
    10
    -
  11  3500
    965
    35
    -
  12  7000
    930
    70
    -
  13  10000
    900
    100
    -
  14  30000
    700
    300
    -
?. [Prepare Standards for 1 mL and 25 mL methods]
Standards forsamples (field samples)
25 mL
?. [Prepare Standards for 1 mL and 25 mL methods]
1ostocks:mixes 1, 2, 3, 4, 5-6, and thiol* (optional). Thiol mix is prepared fresh, if possible.
?. [Prepare Standards for 1 mL and 25 mL methods]
2ostock:  mix of all metabolites. each of the 6 mixes above intototal volume in matrix
80 µl
8 mL
?. [Prepare Standards for 1 mL and 25 mL methods]
3ostock:mix of all metabolites. of secondary stock into  total volume in matrix.
400 µl
8 mL
?. [Prepare Standards for 1 mL and 25 mL methods]
SIL-IS: Prepare a pooled sample by combining filtered, pre-derivatization sub-aliquots of each of the samples. Prepare a  standard mix. Derivatize this with 13C6-benzoyl chloride using the same protocol as for samples and spike into all samples and standards after they have been derivatized. Adding  will result in a final concentration of .
62.5 µl
?. [Prepare Standards for 1 mL and 25 mL methods]
Prepare standards and MQ blanks in duplicate. Due to matrix effects, the MQ blank cannot be used quantitatively as a 'blank' but rather is used to identify contamination (e.g. in the reagents or vials) in a particular sample batch.
?. [Prepare Standards for 1 mL and 25 mL methods]
Volume additions for standards.  ABC1  [Standard] (pg/ml)
  Volume matrix (ml)Vol 3o stock (uL)2  0
    25
    0
  3  5
    25
    25
  4  10
    24.95
    50
  5  30
    24.85
    150
  6  70
    24.65
    350
  7  100
    24.5
    500
  8  300
    23.5
    1500
  9  700
    21.5
    350010  1000
    20
    5000
ABC1  [Standard] (pg/ml)
  Volume matrix (ml)Vol 3o stock (uL)2  0
    25
    0
  3  5
    25
    25
  4  10
    24.95
    50
  5  30
    24.85
    150
  6  70
    24.65
    350
  7  100
    24.5
    500
  8  300
    23.5
    1500
  9  700
    21.5
    350010  1000
    20
    5000
?. [Prepare Reagents]
Prepare reagents.
?. [Prepare Reagents]
Prepare working reagent (  benzoyl chloride in acetone). Avoid plastic (e.g. use glass Hamilton syringes with metal needle instead of pipettes). Prepare fresh daily.
Benzoyl chloride is toxic, flammable, and corrosive. Use under hood with proper PPE.
?. [Prepare Reagents]
Prepare   NaOH. Store indefinitely at room temperature.
?. [Prepare Reagents]
PrepareHCl. Store indefinitely at room temperature in teflon or glass.
?. [Derivatize Samples]
Derivatization procedure for all samples and standards. Follow the same procedure to prepare stable isotope labeled internal standards (SIL-IS), substituting 13C6 benzoyl chloride for the 12C benzoyl chloride. Prior to this method, samples have been filtered (0.2 um, teflon (Omnipore)). Samples should be derivatized as quickly as possible after collection and stored at  if they cannot be processed right away. This recipe is for a  sample. Scale volumes accordingly for the sample size.
-20 °C
1 mL
?. [Derivatize Samples]
AddNaOH () and invert 5 times.
30 µl
?. [Derivatize Samples]
Add working reagent. You will observe "beads" of benzoyl chloride that look "oily", like oil in vinegar salad dressing.
200 µl
?. [Derivatize Samples]
Mix samples for, either by vortexing (volumes ≤ in 8 mL vials and on land) or manually inverting sample rack (larger volumes and at sea).
5 µl
?. [Derivatize Samples]
Add  phosphoric acid. Invert 5 times. The sample will immediately become clear. A white precipitate may form over time .
15 µl
?. [Derivatize Samples]
Add spike of SIL-IS mix to all samples/standards. Samples can now be stored at .
-20 °C
?. [Post-Derivatization Cleanup]
Remove acetone from the samples using either the Vacufuge or a stream of nitrogen gas.
?. [Post-Derivatization Cleanup]
Setup extraction manifold/vaccuum and extract derivatives (1g/6 mL, Bond Elut PPL, Agilent).
?. [Post-Derivatization Cleanup]
Separate precipitate (as best you can) from liquid either by centrifuging (  for ) or waiting for the precipitate to naturally separate. Samples are centrifuged at low speed because the glass vials break at higher speeds. Also, the larger glass vials (EPA 40 mL) sometimes break even at this low speed. These methods do not achieve perfect separation of liquid and solid phases. Better separation could be achieved by transferring samples to centrifuge tubes and centrifuging at a higher speed, but this additional work does not seem to be necessary, and we try to avoid using unnecessary plastics for fear of plasticizer contamination.
Centrifuge: 1000 34, 22 10
?. [Post-Derivatization Cleanup]
Condition PPL cartridge with  methanol (Optima).
6 mL
?. [Post-Derivatization Cleanup]
Rinse PPL cartridge 4 times with   HCl. Don’t let the cartridge dry out.
?. [Post-Derivatization Cleanup]
Carefully remove the id layer (same volume as initial sample volume, e.g. (    or  ) and load onto the PPL cartridge. It is difficult to avoid transferring some precipitate along with the liquid, but try to minimize this. Let the sample drip through the cartridge by gravity or very gentle pressure (
1 mL
25 mL
?. [Post-Derivatization Cleanup]
Rinse the PPL cartridge 4x with  HCl and then dry (pull air through at ~15" Hg) for 5 minutes.
?. [Post-Derivatization Cleanup]
Elute with  methanol (Optima grade) into combusted cuvettes.
6 mL
?. [Post-Derivatization Cleanup]
Transfer eluent to  combusted amber vials. Label caps as well as vials. Store in flammable freezer or proceed to next step.
8 mL
?. [Post-Derivatization Cleanup]
Dry down samples in vacufuge (Eppendorf). Use the alcohol setting unattended for 3.5 hours, and then switch to the aqueous setting until drying is completed, being careful not to over dry. As the samples dry, a white precipitate may form a film on top of the liquid, which can inhibit drying. Periodically tap the vials against a firm surface to break up this film.
?. [Post-Derivatization Cleanup]
Reconstitute samples and remove precipitate. This procedure varies based on sample volume. For  sample volume, complete all steps. For ≤  , complete steps 12.5 to 12.9.
25 mL
5 mL
?. [Post-Derivatization Cleanup]
"Wash" the benzoic acid precipitate out of the samples. Add  acetonitrile/DI to each sample and vortex thoroughly. The precipitate will mostly not dissolve.
500 µl
?. [Post-Derivatization Cleanup]
Centrifuge  for  to separate ppt and liquid.
Centrifuge: 1000 34, 22 10
?. [Post-Derivatization Cleanup]
Transfer the liquid into a  tube.  It is more important to get all the liquid then to avoid transferring some precipitate.
2 mL
?. [Post-Derivatization Cleanup]
Dry down (vacufuge) on aqueous setting with   heat.
30 °C
?. [Post-Derivatization Cleanup]
Add  acetonitrile/DI, vortex thoroughly, and transfer to centrifuge tubes.
100 µl
?. [Post-Derivatization Cleanup]
Centrifuge at  for  .
Centrifuge: 12000 34, 22 10
?. [Post-Derivatization Cleanup]
Transfer liquid to LC vial with appropriate insert.
?. [Post-Derivatization Cleanup]
Add   acetonitrile.
5 µl
?. [Post-Derivatization Cleanup]
Store at . Samples are stored above freezing to minimize precipitation.
4 °C
?. [UPLC-MSMS]
Detection with Ultrahigh Performance Liquid Chromatography (UPLC) Tandem Mass Spectrometry.
?. [Quantify Metabolites]
Use Skyline.